Cloning and recombinant production of CRF receptor(s)

ABSTRACT

In accordance with the present invention, there are provided novel G-protein-coupled receptor proteins (CRF-R) characterized by having sufficient binding affinity for corticotropin releasing factor (CRF) such that concentrations of £ 10 nM of CRF occupy  3 50% of the binding sites of said receptor protein. Nucleic acid sequences encoding such receptors, assays employing same, as well as antibodies derived therefrom, are also disclosed. Invention CRF-Rs can be employed in a variety of ways, such as, for example, in bioassays, for production of antibodies thereto, in therapeutic compositions containing such proteins and/or antibodies.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.08/483,139, filed Jun. 7, 1995, which is a continuation-in-part of U.S.Ser. No. 08/353,537, filed Dec. 9, 1994, now pending, which is acontinuation-in-part of PCT Application No. PCT/US94/05908, filed May25, 1994, now pending, which is a continuation-in-part of U.S. Ser. No.08/110,286, filed Aug. 23, 1993, now pending, which is acontinuation-in-part of U.S. Ser. No. 08/079,320, filed Jun. 18, 1993,now abandoned.

ACKNOWLEDGEMENT

[0002] This invention was made with United States Government supportunder Grant Number DK26745, awarded by the National Institutes ofHealth. The United States Government has certain rights in thisinvention.

FIELD OF THE INVENTION

[0003] The present invention relates to receptor proteins, DNA sequencesencoding same, and various uses therefor.

BACKGROUND OF THE INVENTION

[0004] Corticotropin-releasing factor (CRF) is a 41-residue hypothalamicpeptide which stimulates the secretion and biosynthesis of pituitaryadrenocorticotrophic hormone (ACTH) leading to increased adrenalglucocorticoid production. CRF was originally isolated and characterizedon the basis of its role in this hypothalamic-pituitary-adrenal axis(HPA) [Vale et al., Science Vol. 213:1394-1397 (1981)]. More recently,however, CRF has been found to be distributed broadly within the centralnervous system. (CNS) as well as in extra-neural tissues such as theadrenal glands and testes [Swanson et al., Neuroendocrinology Vol.36:165-186 (1983); Suda et al., J. Clin. Endocrinol. Metab. Vol.58:919-924 (1984; Fabbri and Dufau, Endocrinology Vol. 127:1541-1543(1990)], and sites of inflammation, where it may also act as a paracrineregulator or neurotransmitter.

[0005] In addition to the critical role of CRF in mediating HPA axisactivation, it has been shown to modulate autonomic and behavioralchanges that occur during the stress response. Many of these behavioralchanges have been shown to occur independently of HPA activation in thatthey are insensitive to dexamethasone treatment and hypophysectomy[Britton et al., Life Sci. Vol. 38:211-216 (1986); Britton et al., LifeSci. Vol. 39:1281-1286 (1986); Berridge and Dunn, Pharm. Bioch. Behav.Vol. 34:517-519 (1989)]. In addition, direct infusion of CRF into theCNS mimics autonomic and behavioral responses to a variety of stressors[Sutton et al., Nature Vol. 297:331-333 (1982); Brown and Fisher, BrainRes. Vol. 280:75-79 (1983); Stephens et al., Peptides Vol. 9:1067-1070(1988); Butler et al., J. Neurosci. Vol. 10:176-183 (1990)].Furthermore, peripheral administration of CRF or the CRF antagonist,a-helical CRF 9-41, failed to affect these changes, thus supporting adirect brain action for CRF in such functions. CRF antagonists givenperipherally attenuate stress-mediated increases in ACTH secretion, andwhen delivered into the cerebral ventricles can mitigate stress inducedchanges in autonomic activity and behavior.

[0006] As a result of the extensive anatomical distribution and multiplebiological actions of CRF, this regulatory peptide is believed to beinvolved in the regulation of numerous biological processes. The peptidehas been implicated in the regulation of inflammatory responses. On theone hand, it has been observed that CRF plays a pro-inflammatory role incertain animal models, while in others CRF can suppress inflammation byreducing injury induced increases in vascular permeability.

[0007] It has also been found that CRF can modify steroid production bythe gonads, placenta, and adrenal glands. CRF also has vascular effectssuch as dilating the superior mesenteric arterial bed and dilating thecoronary arteries. In addition to CRF acting on the central nervoussystem to modify gastrointestinal function, CRF has been found todirectly effect the gastrointestinal tract as well.

[0008] In order to more fully investigate the role of CRF within theendocrine, gastrointestinal, reproductive, central nervous and immunesystems, and the possible interactions of CRF with its cognate receptor,it would be desirable to have available a ready source of CRF receptor.Furthermore, the availability of recombinant receptor would allow thedevelopment of less expensive, more sensitive, and automated means forassaying CRF and CRF-like compounds and developing CRF-basedtherapeutics.

[0009] The responsivity to CRF or the quantity of CRF receptors intarget tissues has been shown or predicted (from altered sensitivity toCRF) to change in response to a variety of circumstances includingAlzheimer's Disease, melancholic depression, anorexia nervosa, Cushing'sDisease, alcoholism, and the like. Thus, the development of specificanti-CRF-R antibodies and molecular probes for CRF receptor(s) aredesired for use in appropriate diagnostic assays.

BRIEF DESCRIPTION OF THE INVENTION

[0010] In accordance with the present invention, there are provided newG-protein-coupled receptor proteins which have high binding affinity forcorticotropin-releasing factor (CRF), said proteins are referred tohereinafter as CRF-receptor(s) (CRF-Rs). Invention receptor(s) areprincipal neuroregulators of the hypothalamic-pituitary-adrenal corticalaxis and play an important role in coordinating the endocrine, autonomicand behavioral responses to stress and immune challenge. CRF-Rs arefunctionally coupled to adenylate cyclase as it transduces the signalfor CRF-stimulated intracellular cAMP accumulation. Invention CRF-Rs canbe employed in a variety of ways, such as, for example, in bioassays,for production of antibodies thereto, in therapeutic compositionscontaining such proteins and/or antibodies, and the like.

[0011] In accordance with another aspect of the present invention,binding assays employing CRF-Rs are provided, useful for rapidlyscreening a large number of compounds to determine which compounds(e.g., agonists and antagonists) are capable of binding to the receptorsof the invention. The invention binding assays may also be employed toidentify new CRF-like ligands (e.g., putative mammalian sauvagine orurotensin). Test samples (e.g., biological fluids) may also be subjectedto invention binding assays to detect the presence or absence of CRF orCRF-like compounds.

[0012] In accordance with the present invention, recombinant DNAmolecules encoding CRF-Rs are also provided. DNA molecules encodingCRF-Rs (or fragments thereof) are useful, for example, as probes fordetecting the presence of CRF-R encoding nucleic acids in biologicalsamples, the identification of additional CRF receptor proteins, ascoding sequences which can be used for the recombinant expression of theinvention receptor proteins (or functional fragments thereof), and thelike. Recombinant human CRF-Rs have been expressed in COS cells and bindto CRF and CRF analogs with high affinity. The recombinant production ofCRF-Rs makes feasible their use in the foregoing manners. Fragments ofCRF-R encoding nucleic acid can also be employed as primers for PCRamplification of CRF-R encoding DNA. In addition, sequences derived fromsequences encoding CRF-Rs can also be used in gene therapy applicationsto target the expression of vectors carrying useful genes to specificcell types.

[0013] In accordance with another aspect of the present invention,anti-CRF-R antibodies are also provided. CRF-R and anti-CRF-R antibodiesare useful for diagnostic assays to determine levels of CRF-Rs invarious tissue samples, e.g., neoplastic tissues, and the like.Anti-CRF-R antibodies can also be used to purify CRF-R protein.Moreover, these antibodies are considered therapeutically useful tocounteract or supplement the biological effect of CRF-Rs in vivo.

[0014] Methods and diagnostic systems for determining the levels ofCRF-R in various tissue samples, and levels of CRF-R peptide fragmentsand CRF in vascular fluid samples, are also provided. These diagnosticmethods can be used, for example, for monitoring the level oftherapeutically administered CRF-R (or fragments thereof) to facilitatethe maintenance of therapeutically effective amounts. These diagnosticmethods can also be used to diagnose physiological disorders that resultfrom abnormal levels of CRF or CRF-R.

[0015] CRF-Rs, fragments thereof that bind CRF, or analogs thereof, arecapable of therapeutically modulating the effect of CRF. For example,CRF-R fragments can inhibit CRF binding to CRF-R and can inhibitCRF-induced ACTH release in vitro by pituitary cells. Thus, CRF-Rs canbe administered therapeutically in mammals to reduce high ACTH levelscaused by excess CRF. Such treatments can be used, for example, to treatCushing's Disease, and the like. These CRF-Rs are also useful incombating pituitary tumors that produce CRF. Moreover, they can be usedto reduce pituitary ACTH secretion and hence reduce cortisol levelsunder any condition in which they are abnormally high, such as, forexample, during chronic stress, in patients afflicted with anorexianervosa or alcoholism, and the like. CRF-Rs administered intravenously(IV) are effective to prevent CRF-induced ACTH release. Furthermore, itis contemplated that IV administration of CRF-Rs can reduce intestinaltransit time and thus combat irritable bowel syndrome.

BRIEF DESCRIPTION OF THE FIGURES

[0016]FIG. 1 illustrates the pharmacologic characteristics of plasmidhctCRFR (“human Cushing's Tumor Corticotropin-releasingfactor-receptor”; encoding CRF receptor subtype hCRF-RA₁), transientlyexpressed in COSM6 cells. FIG. 1 presents the results of displacement of¹²⁵I(Nle²¹,Tyr³²) ovine CRF (oCRF) by r/hCRF, when oCRF is bound tomembranes prepared from COSM6 cells transfected with hctCRF receptor(n), or rGnRHR (″), as described in Example 3. The data are from onerepresentative experiment repeated at least four times.

[0017]FIG. 2A illustrates the stimulation of intracellular cAMP in COSM6cells (transfected with plasmid hctCRF, which encodes CRF receptorsubtype CRF-RA₁) by exposure to CRF, hGRF(1-40)OH, VIP, and SalmonCalcitonin, as described in Example 4.

[0018]FIG. 2B illustrates the dose-response stimulation of cAMP in COSM6cells (transfected with plasmid hctCRFR, which encodes CRF receptorsubtype CRF-RA₁) by increasing concentrations of CRF in cells pretreated(n) or untreated (″) with the phosphodiesterase inhibitor, IBMX(3-isobutyl-1-methylxanthine).

[0019]FIG. 2C illustrates the inhibition of CRF stimulated intracellularcAMP by the CRF antagonist a-helical (9-41) CRF. Each determination istaken from a representative experiment performed in triplicate, repeatedat least twice. Cells were pretreated with IBMX. Rat/Human (r/h) CRF wasadded with (solid bars) or without (hollow bars) 2 mM a-helical (9-41).

[0020]FIGS. 3A and 3B illustrate a sequence comparison of mouse CRF-RB(SEQ ID NO:10) with mouse CRF-RA (SEQ ID NO:13). The alignment was madeusing the Jotun-Hein method with PAM250 residue weight table. Putativetransmembrane domains are indicated with a solid bar above the sequence.Potential glycosylation sites are indicated by an (*).

[0021]FIG. 4 illustrates results from a competitive displacement of¹²⁵I-(Nle21,Tyr32)-ovine CRF bound to membranes from COSM6 cellstransfected with CRF-RB₁. “T” indicates “total hormone” and “B”indicates “bound hormone.” The data are pooled from three independentexperiments.

[0022]FIG. 5 illustrates the accumulation of intracellular cAMP in COSM6cells transfected with pCRF-RB₁ stimulated by r/hCRF (n), sauvagine (O),suckerfish urotensin (_), hGRF(1-40) OH (o), and VIP (o). Data are alsoshown for the inhibition of stimulation when the cells are exposed to 1mM antagonist (DPhe¹²,Nle^(21,38))hCRF(12-41) (t). The data are from onerepresentative experiment described in Example 9, repeated at leasttwice. The error bars represent the SEM and are smaller than the symbolsif not visible.

DETAILED DESCRIPTION OF THE INVENTION

[0023] In accordance with the present invention, there is provided afamily of isolated mammalian G-protein-coupled CRF-R proteinscharacterized as having sufficient binding affinity for CRF and CRF-likeligands such that concentrations of £ 10 nM of CRF or CRF-like ligandsoccupy ³50% of the binding sites of approximately 0.8 nM of saidreceptor protein (or approximately 10-20 pmol receptor/mg membraneprotein).

[0024] Use of the phrase “isolated” in the present specification andclaims as a modifier of DNA, RNA, polypeptides or proteins means thatthe DNA, RNA, polypeptides or proteins so designated have been producedin such form by the hand of man, and thus are separated from theirnative in vivo cellular environment. As a result of this humanintervention, the recombinant, isolated and/or substantially pure DNAs,RNAs, polypeptides and proteins of the invention can be produced inlarge quantities and are useful in ways that the DNAs, RNAs,polypeptides or proteins as they naturally occur are not, such asidentification of selective drugs or compounds.

[0025] As used herein, “mammalian” refers to the variety of species fromwhich the invention CRF-R protein is derived, e.g., human, rat, mouse,rabbit, monkey, baboon, bovine, porcine, ovine, canine, feline, and thelike. Invention receptors can be derived from a variety of tissuesources, such as, for example, pituitary cells, placental cells, spleencells, adrenal cells, hematopoietic cells, brain cells, gonadal cells,mesenchymal cells, kidney cells, and the like.

[0026] As employed herein, the term “CRF-R” refers to a family ofisolated and/or substantially pure receptor protein subtypes whichparticipate in the G-protein-coupled response of cells to CRF andCRF-like ligands. Exemplary CRF peptides include r/h CRF and ovine CRF(see U.S. Pat. No. 4,415,558), and the like. As employed herein, thephrase “CRF-like ligands” includes substances which have a substantialdegree of homology (at least 20% homology) with the amino acid sequenceof naturally occurring mammalian CRF, as well as alleles, fragments,homologs or derivatives thereof which have substantially the samebiological activity as mammalian CRF. Suitable CRF-like ligands can beobtained from a variety of vertebrate species and include such compoundsas sauvagine (see, e.g., U.S. Pat. No. 4,605,642), urotensin (see, e.g.,U.S. Pat. Nos. 4,908,352; 4,533,654; and 4,525,189) the CRF analogsdescribed in U.S. Pat. Nos.: 4,415,558; 4,489,163; 4,594,329; 4,605,642;5,109,111, each of which are incorporated herein by reference, and thelike.

[0027] Such receptor subtypes are typically characterized by havingseven putative transmembrane domains, preceded by a large extracellularamino-terminal domain and followed by a large intracellularcarboxy-terminal domain. Hydropathy analysis of exemplary inventionCRF-Rs (described in SEQ ID NOs: 2, 4, 6 and 10) indicates eighthydrophobic regions of approximately 20 amino acids, corresponding to apossible signal peptide at the N-terminus, plus seven putativetransmembrane domains. After removal of the signal peptide, an exemplaryinvention receptor (as described, for example, in SEQ ID NO:2) has amolecular weight of approximately 40-45 kilodaltons.

[0028] Exemplary CRF-R amino acid structures are set forth in SEQ ID NOs2, 4, 6, 8, 10 and 15 of the Sequence Listing provided hereinafter. TheCRF-R described in SEQ ID NO:2 contains five potential glycosylationsites at amino acid positions 38, 45, 78, 90 and 98 (and is referred toherein as CRF-RA₁). Potential protein kinase C phosphorylation sites arelocated in the first and second intracellular loops and in theC-terminal tail at positions 146, 222, 386, and 408. Potential caseinkinase II and protein kinase A phosphorylation sites are located atpositions 301 and 302, respectively. The third intracellular loop of theinvention CRF-R set forth in SEQ ID NO:2 contains an amino acid sequencesimilar to the G_(s) activating region found in the third intracellularloop of the b₂-adrenergic receptor.

[0029] The invention receptor described in SEQ ID NO:2 exhibitsappropriate pharmacologic specificity, i.e., having high affinity forhuman/rat CRF, ovine CRF, the CRF antagonist a helical (9-41) CRF,(DPhe¹²,Nle^(21,38))hCRF(12-41), urotensins, sauvagine, and very lowaffinity for the biologically impotent analog, [Ala¹⁴]-oCRF. A series ofnon-related peptides are inactive, including such compounds as growthhormone releasing factor, salmon calcitonin, vasoactive intestinalpolypeptide, and gonadotropin releasing hormone, as shown in FIG. 2C.

[0030] Binding affinity (which can be expressed in terms of associationconstants, Ka, or dissociation constants, K_(d)) refers to the strengthof interaction between ligand and receptor, and can be expressed interms of the concentration of ligand necessary to occupy one-half (50%)of the binding sites of the receptor. A receptor having a high bindingaffinity for a given ligand will require the presence of very littleligand to become at least 50% bound (hence the K_(d) value will be asmall number); conversely, receptor having a low binding affinity for agiven ligand will require the presence of high levels of ligand tobecome 50% bound (hence the K_(d) value will be a large number).

[0031] Reference to receptor protein “having sufficient binding affinitysuch that concentrations of CRF less or CRF-like peptides than or equalto 10 nM (i.e., £ 10 nM) occupy ³50% (i.e., greater than or equal toone-half) of the binding sites of said receptor protein” means thatligand (i.e., CRF) concentration(s) of no greater than about 10 nM arerequired in order for the ligand to occupy at least 50% of the activesites of approximately 0.8 nM of said receptor (or approximately 10-20pmol receptor/mg membrane protein), with much lower ligandconcentrations typically being required. Presently preferred receptorsare those which have a binding affinity such that ligandconcentration(s) in the range of only about 1-10 nM are required inorder to occupy (or bind to) at least 50% of the receptor binding sites.

[0032] Members of the invention family of receptors can be divided intovarious subclasses, based on the degree of similarity between specificmembers. For example, genomic sequences encoding CRF receptors of thesame subclass typically have substantially similar restriction maps,while genomic sequences encoding CRF receptors of different subclassestypically have substantially different restriction maps. In addition,sequences encoding members of the same subclass of receptors willhybridize under high stringency conditions, whereas sequences encodingmembers of different subclasses will hybridize under low stringencyhybridization conditions, but not under high stringency hybridizationconditions.

[0033] Thus, each member of a given subclass is related to other membersof the same subclass by having a high degree of homology (e.g., >80%overall amino acid homology) between specific members; whereas membersof a given subclass differ from members of a different subclass byhaving a lower degree of homology (e.g., about 30% up to 80% overallamino acid homology) between specific members of different subclasses.

[0034] Based on the above criteria, the receptor species describedherein can be designated as CRF-RA or CRF-RB subtypes. Thus, thereceptor described in SEQ ID NO:2 is a CRF-RA subtype, and is referredto herein as hCRF-RA₁ (for human CRF-R, subtype A, variant 1). Themodified form of hCRF-RA₁ which contains the insert sequence set forthin SEQ ID NO:4 is referred to herein as hCRF-RA₂. Similarly, thereceptor described in SEQ ID NO:6 is referred to herein as rCRF-RA (forrat CRF-R, subtype A), and the receptor described in SEQ ID NOs:8 and 10are referred to herein as mCRF-RB₁ (for mouse CRF-R, subtype B, variant1).

[0035] The mouse CRF-RA₁ and CRF-RB₁ receptors have been compared andare seen to be about 70% homologous at the nucleotide level and 68%homologous at the amino acid level (see, e.g., FIGS. 3A and 3B). Inaddition, there are a number of fundamental structural characteristicsthat are conserved between the two receptors. The number and location ofpotential N-glycosylation sites are the same. Six cysteines are presentin the N-terminal domain, characteristic of the receptor family. In theCRF-RB₁ receptor, however, there are two additional cysteines, one inthe N-terminus and the other at the junction of the first extracellularloop (i.e., ECL-1) and the third transmembrane domain (i.e., TMD-3). Ifone assumes that the N-terminal cysteine is removed with the signalpeptide and that the latter cysteine is within the transmembrane domain,CRF-RB₁ is seen to have six cysteines in the extracellular region, as isthe case for other members of the receptor family.

[0036] The first intracellular loop is practically the same betweenCRF-RA₁ and CRF-RB₁, except for the substitution of a valine for thearginine found in CRF-RA₁. The second intracellular loop differs inthree amino acids, but the changes are conservative. Thus, a methionine,a glutamic acid and a histidine are present in CRF-RB₁, instead of aleucine, an aspartic acid and an arginine, respectively, in CRF-RA₁. Thethird intracellular loop is 100% identical between the two receptors.The C-terminal domain is also highly conserved between the tworeceptors. The putative phosphorylation sites in the intracellular loopsare also nearly identical between CRF-RA₁ and CRF-RB₁, with oneexception occurring in the C-terminus, in which SER386 in CRF-RA₁ ispresent as an alanine in CRF-RB₁.

[0037] A major determinant of the coupling invention CRF-receptors toGTP-binding proteins and subsequently to adenylate cyclase is thought toreside in the third intracellular loop and the C-terminus. Because ofthe high similarity of the third intracellular loop and the C-termini ofCRF-RA₁ and CRF-RB₁, the coupling and signal transduction properties ofthe two receptors are expected to be very similar. Indeed, the data(FIG. 5) demonstrate that the signal transduction characteristics ofCRF-RA₁ and CRF-RB₁ are nearly identical. It is expected that a moredetailed analysis of the desensitization characteristics of the tworeceptors will reveal subtle differences as a consequence of the changesin the C-terminus and the other intracellular loops.

[0038] The main differences between the two receptors are found in theN-terminal domain, in which sixteen extra amino acids are found inCRF-RB₁, and in which there are significant, non-conservative amino acidchanges in the remaining portion of the N-terminus. It is interesting tonote that, based on the genomic sequence of the mouse CRF-RA₁, the aminoacid sequences of the two receptors start to diverge very close to thesecond intron/exon junction in the N-terminus, raising the possibilitythat some of the divergence between the two receptors could result fromalternative exon utilization (i.e., splice variants). Indeed, thepresence of multiple protected RNA species when using N-terminal probesis consistent with the existence of splice variants of this receptor.

[0039] In addition to the N-terminal domain, the first, second, andthird extracellular loops (ECLs-1, -2, and -3) also contain significantdifferences. For example, extracellular loop-1 in CRF-RB₁ contains morecharged residues than does the corresponding loop in CRF-RA₁.Extracellular loop-2 in CRF-RA₁ contains an arginine, instead of aglutamic acid in CRF-RB₁. Extracellular loop-3 is the most similarbetween the two receptors. It is presently believed that a major bindingdeterminant in this family of receptors is the N-terminal region and theextracellular loops. Therefore, the existence of differences in theextracellular domains suggests that the binding specificities betweenthe two receptors should differ. Urotensin (K_(i)=0.7±0.3, n=3) andsauvagine (K_(i)=0.6±0.1, n=3) show a trend to be more potent thanr/hCRF (K_(i)=1.3±0.2, n=6) on CRF-RB.

[0040] In situ hybridization studies indicate that mRNAs related toCRF-RB have a restricted distribution in the central nervous system thatdiffers considerably from that of CRF-RA. Thus, the receptors derivedfrom the two genes, CRF-RA and CRF-RB, with their distinct tissuedistributions and structural diversity especially in the extracellulardomains are likely to subserve disparate biological roles.

[0041] It is expected that a more detailed pharmacological comparison ofthe two receptors is likely to reveal significant differences in thebinding characteristics of the two receptors. It is also expected thatdifferent CRF-R subtypes will mediate different actions of CRF. Thus, byhaving available nucleic acid encoding various CRF-R subtypes, those ofskill in the art have been enabled to screen for and develop selectiveanalogs specific for each CRF-R subtype. The analogs so obtained will bemore specific, potent, and effective at binding and modulating theactivity of the respective CRF-R subtype.

[0042] In one embodiment of the present invention, the CRF-RA₁ encodedby the clone referred to herein as “hctCRFR” (described hereinafter) hasa high binding affinity for r/h CRF [K_(d)=3.3±0.45 nM (n=4)]; ovine CRF[K_(d)=2.3±0.66 nM (n=3)]; and for the antagonist a helCRF(9-41)[K_(d)=13.0±5.2 nM (n=3)]. This receptor has a low binding affinity forthe biologically impotent analog, [Ala¹⁴]-ovine CRF [K_(d)>300 nM (n=2)]. In another embodiment of the present invention, the CRF-R describedin SEQ ID NO:2 has a binding affinity for r/h CRF of K_(d)=3.8±0.20 nM,(n=1).

[0043] Presently preferred receptor proteins of the invention have aminoacid sequences that are substantially the same as the sequences setforth in Sequence ID Nos. 2, 4, 6, 8, and 10 and amino acid sequenceswhich are substantially the same as the amino acid sequences encoded bythe CRF-RA₁-encoding portion of clone hctCRFR, deposited with the ATCCunder accession number 75474, as well as functional, modified formsthereof. Those of skill in the art recognize that numerous residues ofthe above-described sequences can be substituted with other, chemically,sterically and/or electronically similar residues without substantiallyaltering the biological activity of the resulting receptor species.

[0044] The htcCRFR clone was deposited Jun. 2, 1993, at the AmericanType Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.,U.S.A. 20852, under the terms of the Budapest Treaty on theInternational Recognition of Deposits of Microorganisms for Purposes ofPatent Procedure and the Regulations promulgated under this Treaty.Samples of the deposited material are and will be available toindustrial property offices and other persons legally entitled toreceive them under the terms of the Treaty and Regulations and otherwisein compliance with the patent laws and regulations of the United Statesof America and all other nations or international organizations in whichthis application, or an application claiming priority of thisapplication, is filed or in which any patent granted on any suchapplication is granted. In particular, upon issuance of a U.S. patentbased on this or any application claiming priority to or incorporatingthis application by reference thereto, all restriction upon availabilityof the deposited material will be irrevocably removed.

[0045] As employed herein, the term “substantially the same amino acidsequence” refers to amino acid sequences having at least about 70%identity with respect to the reference amino acid sequence, andretaining comparable functional and biological properties characteristicof the protein defined by the reference amino acid sequence. Preferably,proteins having “substantially the same amino acid sequence” will haveat least about 80%, more preferably 90% amino acid identity with respectto the reference amino acid sequence; with greater than about 95% aminoacid sequence identity being especially preferred.

[0046] Recombinant CRF-R protein can be routinely obtained, employingthe invention nucleic acids described hereinafter, having significantlyhigher purity than naturally occurring CRF-R (e.g., substantially freeof other proteins present in crude extracts from mammalian cells).Recombinant DNA techniques well-known in the art, for example, can beused to generate organisms or cell lines that produce heterologous CRF-Rprotein in significantly higher purities, relative to naturallyoccurring membrane protein. Subsequently, using appropriate isolationtechniques, it is possible to routinely obtain CRF-R proteins which areat least about 70%, preferably 80%, more preferably 90%, and mostpreferred 98% pure (by weight of total proteins), and which is hereinreferred to as substantially pure.

[0047] In accordance with a further embodiment of the present invention,there is provided a binding assay employing receptors of the invention,whereby a large number of compounds can be rapidly screened to determinewhich compounds, if any, are capable of binding to the receptors of theinvention. Subsequently, more detailed assays can be carried out withinitially identified compounds, to further determine whether suchcompounds act as agonists or antagonists of invention receptors.

[0048] Another application of the binding assay of the invention is theassay of test samples (e.g., biological fluids) for the presence orabsence of CRF. Thus, for example, serum from a patient displayingsymptoms thought to be related to over- or under-production of CRF canbe assayed to determine if the observed symptoms are indeed caused byover- or under-production of CRF (or CRF receptor).

[0049] The binding assays contemplated by the present invention can becarried out in a variety of ways, as can readily be identified by one ofskill in the art. For example, competitive binding assays can beemployed, as well as radioimmunoassays, ELISA, ERMA, and the like.

[0050] In accordance with a still further embodiment of the presentinvention, there are provided bioassays for evaluating whether testcompounds are capable of acting as agonists or antagonists ofreceptor(s) of the present invention (or functional modified formsthereof).

[0051] Invention CRF-Rs are coupled by heterotrimeric G-proteins tovarious intracellular enzymes, ion channels, and transporters. TheG-proteins associate with invention CRF-R proteins at the intracellularface of the plasma membrane. An agonist binding to CRF-R catalyzes theexchanges of GTP for GDP on the a-subunit (G-protein “activation”),resulting in its dissociation and stimulation of one (or more) of thevarious signal-transducing enzymes and channels. The different G-proteina-subunits preferentially stimulate particular effectors. Thespecificity of signal transduction may be determined, therefore, by thespecificity of G-protein coupling.

[0052] It has been found that invention CRF-R proteins mediate signaltransduction through the modulation of adenylate cyclase. For example,when CRF binds to CRF-R, adenylate cyclase causes an elevation in thelevel of intracellular cAMP. Accordingly, in one embodiment of thepresent invention, the bioassay for evaluating whether test compoundsare capable of acting as agonists or antagonists comprises:

[0053] (a) culturing cells containing:

[0054] DNA which expresses CRF receptor protein(s) or functionalmodified forms thereof,

[0055] wherein said culturing is carried out in the presence of at leastone compound whose ability to modulate signal transduction activity ofCRF receptor protein is sought to be determined, and thereafter

[0056] (b) monitoring said cells for either an increase or decrease inthe level of intracellular cAMP.

[0057] Methods well-known in the art that measure intracellular levelsof cAMP, or measure cyclase activity, can be employed in binding assaysdescribed herein to identify agonists and antagonists of the CRF-R. Forexample, because activation of some G-protein-coupled receptors resultsin decreases or increases in cAMP, assays that measure intracellularcAMP levels (see, e.g., Example 4) can be used to evaluate recombinantCRF-Rs expressed in mammalian host cells.

[0058] As used herein, “ability to modulate signal transduction activityof CRF receptor protein” refers to a compound that has the ability toeither induce or inhibit signal transduction activity of the CRFreceptor protein.

[0059] In another embodiment of the present invention, the bioassay forevaluating whether test compounds are capable of acting as agonistscomprises:

[0060] (a) culturing cells containing:

[0061] DNA which expresses CRF receptor protein(s) or functionalmodified forms thereof, and

[0062] DNA encoding a reporter protein, wherein said DNA is operativelylinked to a CRF-R responsive transcription element;

[0063] wherein said culturing is carried out in the presence of at leastone compound whose ability to induce signal transduction activity of CRFreceptor protein is sought to be determined, and thereafter

[0064] (b) monitoring said cells for expression of said reporterprotein.

[0065] In another embodiment of the present invention, the bioassay forevaluating whether test compounds are capable of acting as antagonistsfor receptor(s) of the invention, or functional modified forms of saidreceptor(s), comprises:

[0066] (a) culturing cells containing:

[0067] DNA which expresses CRF receptor protein(s), or functionalmodified forms thereof, and

[0068] DNA encoding a reporter protein, wherein said DNA is operativelylinked to a CRF-R responsive transcription element

[0069] wherein said culturing is carried out in the presence of:

[0070] increasing concentrations of at least one compound whose abilityto inhibit signal transduction activity of CRF receptor protein(s) issought to be determined, and

[0071] a fixed concentration of at least one agonist for CRF receptorprotein(s), or functional modified forms thereof; and thereafter

[0072] (b) monitoring in said cells the level of expression of saidreporter protein as a function of the concentration of said compound,thereby indicating the ability of said compound to inhibit signaltransduction activity.

[0073] In step (a) of the above-described antagonist bioassay, culturingmay also be carried out in the presence of:

[0074] fixed concentrations of at least one compound whose ability toinhibit signal transduction activity of CRF receptor protein(s) issought to be determined, and

[0075] an increasing concentration of at least one agonist for CRFreceptor protein(s), or functional modified forms thereof.

[0076] Host cells for functional recombinant expression of CRF-Rspreferably express endogenous or recombinant guanine nucleotide-bindingproteins (i.e., G-proteins). G-proteins are a highly conserved family ofmembrane-associated proteins composed of a, b and g subunits. The asubunit, which binds GDP and GTP, differs in different G-proteins. Theattached pair of b and g subunits may or may not be unique; different achains may be linked to an identical bg pair or to different pairs[Linder and Gilman, Sci. Am. 267:56-65 (1992)]. More than 30 differentcDNAs encoding G protein a subunits have been cloned [Simon et al.,Science 252:802 (1991)]. At least four different b polypeptide sequencesare known [Simon et al., Science 252:802 (1991)]. G-proteins switchbetween active and inactive states by guanine nucleotide exchange andGTP hydrolysis. Inactive G protein is stimulated by a ligand-activatedreceptor to exchange GDP for GTP. In the active form, the a subunit,bound to GTP, dissociates from the bg complex, and the subunits theninteract specifically with cellular effector molecules to evoke acellular response. Because different G-proteins can interact withdifferent effector systems (e.g., phospholipase C, adenyl cyclasesystems) and different receptors, it is useful to investigate differenthost cells for expression of different recombinant CRF-R receptorsubtypes. Alternatively, host cells can be transfected with G-proteinsubunit-encoding DNAs for heterologous expression of differing Gproteins.

[0077] Host cells contemplated for use in the bioassay(s) of the presentinvention include CV-1 cells, COS cells, and the like; reporter andexpression plasmids employed typically also contain the origin ofreplication of SV-40; and the reporter and expression plasmids employedalso typically contain a selectable marker.

[0078] As used herein, a “CRF-R responsive transcription element” is anypromoter region that is induced, e.g., by the well-known G-proteinmediated signal transduction mechanism, to initiate transcription uponthe binding of a CRF-R agonist, such as CRF. A preferred CRF-Rresponsive transcription element is a cAMP responsive transcriptionelement. Cyclic AMP (cAMP) responsive transcription elements employed inthe bioassay(s) of the present invention are well-known to those ofskill in the art. The cAMP response elements respond to increases inintracellular cAMP by initiating trascription of the DNA molecule (i.e.,a reporter gene) operatively linked thereto. An exemplary cAMP responseelement suitable for use herein is the human DNA b-Polymerase genepromoter (see Mamula et al., DNA and Cell Bio., 11:61-70, 1992).

[0079] Reporter proteins suitable for use herein are well known in theart. Host cells can be monitored for the level of expression of areporter gene encoding a reporter protein in a variety of ways, such as,for example, by photometric means, e.g., by colorimetry (with a coloredreporter product such as β-galactosidase), by fluorescence (with areporter product such as luciferase), by enzyme activity, and the like.

[0080] Compounds contemplated for screening in accordance with theinvention bioassays include CRF or CRF-like ligands, as well ascompounds which bear no particular structural or biological relatednessto CRF. Suitable compounds may be obtained from well-known sources,e.g., from peptide libraries, chemical libraries, bacterial and yeastbroths, plants, and the like.

[0081] Examples of compounds which bear no particular structural orbiological relatedness to CRF, but which are contemplated for screeningin accordance with the bioassays of the present invention, include anycompound that is an antagonist (i.e., is capable of blocking the actionof the invention receptor peptides), or an agonist (i.e., is capable ofpromoting the action of the invention receptor peptides), such as, forexample, alkaloids and other heterocyclic organic compounds, and thelike.

[0082] As employed herein, the term “non-CRF-like” proteins refers toany organic molecule having essentially no structural similarity withCRF (as defined broadly herein).

[0083] Also encompassed by the term CRF-R are the various subtypesthereof (e.g., CRF-RA (such as hCRF-RA₁, and hCRF-RA₂), CRF-RB₁, and thelike), as well as polypeptide fragments or analogs thereof. Therefore, aCRF-R contemplated by the present invention can be subject to variouschanges, substitutions, insertions, and deletions, where such changesprovide for certain advantages in its use. For example, a peptidefragment is capable of immunologically mimicking a CRF-R nativeantigenic epitope or is capable of exhibiting another biologicalproperty characteristic of CRF-R, such as, for example, binding to CRFor binding to G-protein(s).

[0084] Specific CRF-R residues or regions which are necessary forefficient signal transduction may interact with conserved G-proteinmotifs. In addition, certain short amino acid stretches of the CRF-R,which are necessary for G-protein coupling, also determine thespecificity of the G-protein interactions. Thus, polypeptide fragmentsof the invention CRF-R are useful in assays or therapeutic methods inwhich controlled binding to various G-proteins is desired.

[0085] The term “analog” includes any polypeptide having an amino acidresidue sequence substantially identical to a sequence specificallyshown herein in which one or more residues have been conservativelysubstituted with a functionally similar residue and which displays theability to mimic CRF-R as described herein. Examples of conservativesubstitutions include the substitution of one non-polar (hydrophobic)residue such as isoleucine, valine, leucine or methionine for another,the substitution of one polar (hydrophilic) residue for another such asbetween arginine and lysine, between glutamine and asparagine, betweenglycine and serine, the substitution of one basic residue such aslysine, arginine or histidine for another, or the substitution of oneacidic residue, such as aspartic acid or glutamic acid for another.

[0086] The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residue,provided that such polypeptide displays the requisite binding activity.

[0087] “Chemical derivative” refers to a subject polypeptide having oneor more residues chemically derivatized by reaction of a functional sidegroup. Such derivatized molecules include, for example, those moleculesin which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as chemical derivatives are those peptides which containone or more naturally occurring amino acid derivatives of the twentystandard amino acids. For examples: 4-hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Polypeptides of the present invention also include any polypeptidehaving one or more additions and/or deletions of residues, relative tothe sequence of a polypeptide whose sequence is shown herein, so long asthe requisite activity is maintained.

[0088] When additional residues have been added at either terminus forthe purpose of providing a “linker” by which the polypeptides of theinvention can be conveniently affixed to a label or solid matrix, orcarrier, the linker residues do not form CRF-R epitopes, i.e., are notsimilar in structure to CRF-R. Labels, solid matrices, and carriers thatcan be used with the polypeptides of this invention are describedhereinbelow.

[0089] Amino acid residue linkers include at least one residue up to 40or more residues (more often they comprise 1 to 10 residues), but do notform CRF-R epitopes. Typical amino acid residues used for linking aretyrosine, cysteine, lysine, glutamic acid and aspartic acid. Inaddition, a subject polypeptide can differ in sequence, unless otherwisespecified, from the natural sequence of CRF-R by modification of thesequence by N-terminal acylation e.g., acetylation or thioglycolic acidamidation, and by C-terminal amidation, e.g., with ammonia, methylamine,and the like.

[0090] CRF-R polypeptides of the present invention are capable ofinducing antibodies that immunoreact with CRF-R. In view of the wellestablished principle of immunologic cross-reactivity, the presentinvention therefore contemplates antigenically related variants of thepolypeptides. An “antigenically related variant” is a subjectpolypeptide that is capable of inducing antibody molecules thatimmunoreact with the CRF-R polypeptides described herein.

[0091] CRF-R polypeptides of the present invention can be synthesized byany of the techniques that are known to those skilled in the polypeptideart, including recombinant DNA techniques. Synthetic chemistrytechniques, such as solid-phase Merrifield-type synthesis, are preferredfor producing polypeptide fragments for reasons of purity, antigenicspecificity, freedom from undesired side products, ease of production,and the like. An excellent summary of the many techniques available canbe found in J. M. Steward and J. D. Young, “Solid Phase PeptideSynthesis”, W. H. Freeman Co., San Francisco, 1969; M. Bodansky, et al.,“Peptide Synthesis”, John Wiley & Sons, Second Edition, 1976 and J.Meienhofer, “Hormonal Proteins and Peptides”, Vol. 2, p. 46, AcademicPress (New York), 1983 for solid phase peptide synthesis, and E.Schroder and K. Kubke, “The Peptides”, Vol. 1, Academic Press (NewYork), 1965 for classical solution synthesis, each of which isincorporated herein by reference. Appropriate protective groups usablein such synthesis are described in the above texts and in J. F. W.McOmie, “Protective Groups in Organic Chemistry”, Plenum Press, NewYork, 1973, which is incorporated herein by reference. See also U.S.Pat. No. 5,055,396, incorporated herein by reference.

[0092] CRF-R polypeptides can be used, inter alia, in diagnostic methodsand systems according to the present invention to detect the level ofCRF-R (or fragments thereof) present in a body sample, to detect thelevel of CRF in a body sample, or to prepare an inoculum as describedherein for the preparation of antibodies that immunoreact with epitopeson CRF-R. CRF-R polypeptides can also be used to bind, detect and purifyvarious intracellular G-proteins and CRF-like receptoragonist/antagonists, such as heterocyclic compounds, and the like. Inaddition, CRF-R polypeptides can be used in therapeutic methodsdescribed herein, e.g., to inhibit the CRF-induced ACTH release anddecrease the level of ACTH in a patient.

[0093] In accordance with yet another embodiment of the presentinvention, there are provided antibodies generated against theabove-described receptor proteins. Such antibodies can be employed fordiagnostic applications, therapeutic applications, and the like.Preferably, for therapeutic applications, the antibodies employed willbe monoclonal antibodies.

[0094] The above-described antibodies can be prepared employing standardtechniques, as are well known to those of skill in the art, usinginvention receptor proteins, or fragments thereof, as antigens forantibody production. Antibodies of the present invention are typicallyproduced by immunizing a mammal with an inoculum containing a CRF-Rprotein or fragment thereof thereby inducing the production of antibodymolecules having immunospecificity for the immunizing agent.

[0095] For example, antibodies raised in rabbits against a syntheticpeptide fragment of the invention protein recognize the syntheticpeptide and the corresponding invention CRF-R on an equimolar basis, andpreferably, are capable of inhibiting the activity of the nativeprotein. Antibodies to CRF-R may be obtained, for example, by immunizingthree month old male and female white New Zealand rabbits with asuitable synthetic peptide fragment to which Tyr has been added at theC-terminus in order to couple it, as an antigen, to BSA by abisdiazotized benzidine (BDB) linkage by reaction for 2 hours at 4° C.The reaction mixture is dialyzed to remove low molecular weightmaterial, and the retentate is frozen in liquid nitrogen and stored at−20° C. Animals are immunized with the equivalent of 1 mg of the peptideantigen according to the procedure of Vaughan et al., Meth. inEnzymology, 168:588-617 (1989). At four week intervals, the animals areboosted by injections of 200 mg of the antigen and bled ten to fourteendays later. After the third boost, antiserum is examined for itscapacity to bind radioiodinated antigen peptide prepared by thechloramine-T method and then purified by CMC-ion exchange columnchromatography or HPLC. The antibody molecules are then collected fromthe mammal and isolated to the extent desired by well known techniquessuch as, for example, by using DEAE Sephadex to obtain the IgG fraction.

[0096] To enhance the specificity of the antibody, the antibodies may bepurified by immunoaffinity chromatography using solid-phase immunizingpolypeptide. The antibody is contacted with the solid-phase immunizingpolypeptide for a period of time sufficient for the polypeptide toimmunoreact with the antibody molecules to form a solid-phaseimmunocomplex. The bound antibodies are separated from the complex bystandard techniques.

[0097] Antibody so produced can be used, inter alia, in diagnosticmethods and systems to detect the level of CRF-R present in a mammalian,preferably human, body sample, such as tissue or vascular fluid. Theanti-CRF-R antibodies can also be used for immunoaffinity or affinitychromatography purification of CRF-R biological materials. In addition,an anti-CRF-R antibody according to the present invention can be used inmammalian therapeutic methods, preferably human, as a CRF-R agonist orantagonist, to neutralize or modulate the effect of CRF-R, increase thelevel of free CRF (e.g., CRF not bound by CRF-R), increase CRF-inducedACTH release, increase the level of ACTH-induced glucocorticoids in apatient, and the like.

[0098] The proteins of the invention, and the antibodies of theinvention, can be administered to a subject employing standard methods,such as, for example, by intraperitoneal, intramuscular, intravenous, orsubcutaneous injection, and the like. Implant and transdermal modes ofadministration are also appropriate. In addition, proteins of theinvention can be delivered by transfection with viral or retroviralvectors that encode invention protein. One of skill in the art canreadily determine dose forms, treatment regiments, etc, depending on themode of administration employed.

[0099] In accordance with a further embodiment of the present invention,there are provided isolated and purified nucleic acid molecules (e.g.,DNA or RNA) which encode the above-described receptor proteins. Thenucleic acid molecules described herein are useful for producinginvention CRF-R proteins, when such nucleic acids are incorporated intoa variety of protein expression systems known to those of skill in theart. In addition, such nucleic acid molecules (or fragments thereof) canbe labeled with a readily detectable substituent and used ashybridization probes for assaying for the presence and/or amount of aCRF-R gene or mRNA transcript in a given sample. Such nucleic acidmolecules (or fragments thereof), when labeled with a readily detectablesubstituent, can also be used as hybridization probes for identifyingadditional CRF-R genes. The nucleic acid molecules described herein, andfragments thereof, are also useful as primers and/or templates in a PCRreaction for amplifying genes encoding the CRF-R protein describedherein. In addition, the nucleic acid molecules described herein, andfragments thereof, are also useful as primers and/or templates in a PCRreaction for identifying genes encoding additional CRF-R proteins whichare part of the family of receptor proteins described herein.

[0100] The above-described receptor(s) can be encoded by numerousnucleic acid molecules, e.g., a nucleic acid molecule having acontiguous nucleotide sequence substantially the same as:

[0101] nucleotides 82-1329 of Sequence ID No. 1,

[0102] nucleotides 82-1329 of Sequence ID No. 1, further containingnucleotides 1-87 of SEQ ID No. 3 inserted between nucleotides 516-517 ofSEQ ID No. 1,

[0103] nucleotides 81-1324 of Sequence ID No. 5,

[0104] substantially all nucleotides of Sequence ID No. 7,

[0105] nucleotides 79-1371 of Sequence ID No. 9,

[0106] the CRF-RA₁-encoding portion of clone hctCRFR, deposited with theATCC under accession number 75474,

[0107] or variations thereof which encode the same amino acid sequences,but employ different codons for some of the amino acids,

[0108] or splice variant cDNA sequences thereof.

[0109] As employed herein, the phrase “nucleic acid” refers toribonucleic acid (RNA) or deoxyribonucleic acid (DNA). DNA can be eithercomplementary DNA (cDNA) or genomic DNA, e.g. a gene encoding a CRF-R.

[0110] As employed herein, the phrases “contiguous nucleotide sequencesubstantially the same as” or “substantially the same nucleotidesequence” refers to DNA having sufficient homology to the referencepolynucleotide, such that it will hybridize to the reference nucleotideunder typical moderate stringency conditions. In one embodiment, nucleicacid molecules having substantially the same nucleotide sequence as thereference nucleotide sequence encodes substantially the same amino acidsequence as that of any one of SEQ ID NOs:2, 4, 6, 8, or 10. In anotherembodiment, DNA having “substantially the same nucleotide sequence” asthe reference nucleotide sequence has at least 60% homology with respectto the reference nucleotide sequence. DNA having at least 70%, morepreferably 80%, yet more preferably 90%, homology to the referencenucleotide sequence is preferred.

[0111] Yet other DNAs which encode the above-described receptor arethose having a contiguous nucleotide sequence substantially the same asset forth in Sequence ID Nos. 1, 3, 5, 7, or 9 or the CRF-RA₁-encodingportion of clone hctCRFR, deposited with the ATCC under accession number75474.

[0112] “Gene(s)” (i.e., genomic DNA) encoding invention CRF-Rs typicallycontain at least two introns. Thus, alternatively spliced variant cDNAsequences encoding invention CRF-Rs are contemplated herein (e.g.,CRF-RA₂). For example, SEQ ID NO:3 sets forth an 87 bp cDNA splicevariant insert sequence that is inserted between nucleotide positions516-517 of the CRF-RA₁, encoding cDNA set forth in SEQ ID NO:1 (therebyproducing CRF-RA₂).

[0113] As used herein, the phrases “splice variant” or “alternativelyspliced”, when used to describe a particular nucleotide sequenceencoding an invention receptor, refers to a cDNA sequence that resultsfrom the well known eukaryotic RNA splicing process. The RNA splicingprocess involves the removal of introns and the joining of exons fromeukaryotic primary RNA transcripts to create mature RNA molecules of thecytoplasm.

[0114] Methods of isolating splice variant nucleotide sequences are wellknown in the art. For example, one of skill in the art can employnucleotide probes derived from the CRF-R encoding cDNA of SEQ ID NOs 1,3, 5, 7, or 9 to screen the Cushing's tumor cDNA library described inthe Examples or other cDNA libraries derived from cells believed toexpress CRF-Rs, e.g., brain, heart, pituitary, immune, gonadal, adrenal,placental, gastrointestinal, pulmonary, corticotropic, and the like.

[0115] In a preferred embodiment, cDNA encoding CRF-Rs as disclosedherein have substantially the same nucleotide sequence as nucleotides82-1329 of SEQ ID NO:1, as nucleotides 82-1329 of SEQ ID NO:1 furthercontaining nucleotides 1-87 of SEQ ID NO:3 inserted between nucleotides516-517 of SEQ ID NO:1, as SEQ ID NO:5, as nucleotides 81-1324 of SEQ IDNO:7, or as nucleotides 79-1371 of SEQ ID NO:9. The presently mostpreferred cDNA molecules encoding the CRF-Rs have the same nucleotidesequence as nucleotides 82-1329 of SEQ ID NO:1, as nucleotides 82-1329of SEQ ID NO:1 further containing nucleotides 1-87 of SEQ ID NO:3inserted between nucleotides 516-517 of SEQ ID NO:1, as SEQ ID NO:5, asnucleotides 81-1324 of SEQ ID NO:7, or as nucleotides 79-1371 of SEQ IDNO:9.

[0116] In accordance with another embodiment of the present invention,isolated and purified nucleic acid encoding a CRF-R may be selectedfrom:

[0117] (a) DNA encoding the amino acid sequence set forth in SEQ IDNO:2, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10; or DNA encoding theamino acid sequence set forth in SEQ ID NO:2 further comprising theamino acid sequence set forth in SEQ ID NO:4 inserted between aminoacids 145-146 of SEQ ID NO:2, or

[0118] (b) DNA that hybridizes to the DNA of (a) under moderatelystringent conditions, wherein said DNA encodes biologically activeCRF-R, or

[0119] (c) DNA degenerate with respect to either (a) or (b) above,wherein said DNA encodes biologically active CRF-R.

[0120] Hybridization refers to the binding of complementary strands ofnucleic acid (i.e., sense:antisense strands or probe:target-DNA) to eachother through hydrogen bonds, similar to the bonds that naturally occurin chromosal DNA. Stringency levels used to hybridize a given probe withtarget-DNA can be readily varied by those of skill in the art.

[0121] As used herein, the phrase “moderately stringent” hybridizationrefers to conditions that permit target-DNA to bind a complementarynucleic acid that has about 60%, preferably about 75%, more preferablyabout 85%, homology to the target DNA; with greater than about 90%homology to target-DNA being especially preferred. Preferably,moderately stringent conditions are conditions equivalent tohybridization in 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDSat 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 65° C.Denhart's solution and SSPE (see, e.g., Sambrook et al., MolecularCloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989)are well known to those of skill in the art as are other suitablehybridization buffers.

[0122] The term “functional” or “biologically active”, when used hereinas a modifier of receptor protein(s) of the present invention, refers toa polypeptide that is able to produce one of the functionalcharacteristics, e.g., antigenicity, exhibited by any of the CRF-Rsdescribed herein. In another embodiment, biologically active means thatbinding of CRF-like ligands (such as CRF analogs, urotensin, sauvagine,and the like) to said receptor protein(s) modifies the receptorinteraction with G-proteins, which in turn affects the levels ofintracellular second messengers, preferably cAMP, leading to a varietyof physiological effects. Stated another way, “functional” means that asignal is transduced as a consequence of agonist activation of receptorprotein(s).

[0123] As used herein, the term “degenerate” refers to codons thatdiffer in at least one nucleotide from a reference nucleic acid, e.g.,SEQ ID NO:1, but encode the same amino acids as the reference nucleicacid. For example, codons specified by the triplets “UCU”, “UCC”, “UCA”,and “UCG” are degenerate with respect to each other since all four ofthese codons encode the amino acid serine.

[0124] The invention nucleic acids can be produced by a variety ofmethods well-known in the art, e.g., the methods described in Examples 1and 5, employing PCR amplification using oligonucleotide primers fromvarious regions of SEQ ID NOs:1, 3, 5, 7, 9 and 14 and the like.

[0125] One method employed for isolating and cloning nucleic acidsencoding the receptor(s) of the present invention involves expressing,in mammalian cells, a cDNA library prepared from any cell type thoughtto respond to CRF (e.g., pituitary cells, placental cells, fibroblastcells, and the like) in a suitable host cell, such as, for example,COSM6 cells. The ability of the resulting mammalian cells to bind alabeled receptor ligand (i.e., a labeled CRF analog) is then determined.Finally, the desired cDNA insert(s) are recovered, based on the abilityof a particular cDNA, when expressed in mammalian cells, to induce (orenhance) the binding of labeled receptor ligand to said cell.

[0126] Alternatively, DNA libraries may be screened employing animmunological expression assay with an antibody raised against theprotein of interest. Screening of the expression library with antibodiesraised against the protein of interest may also be used, alone or inconjunction with hybridization probing, to identify or confirm thepresence of the sought-after DNA sequences in DNA library clones. Suchtechniques are taught, for example, in Maniatis et al., Cold SpringHarbor Laboratory Manual, Cold Spring Harbor, N.Y. (1982), (hereinafterCSH).

[0127] In accordance with a further embodiment of the present invention,optionally labeled receptor-encoding cDNAs, or fragments thereof, can beemployed to probe library(ies) (e.g., cDNA, genomic, and the like) foradditional nucleotide sequences encoding novel mammalian members of theCRF receptor family. Such screening is initially carried out underlow-stringency conditions, which comprise a temperature of less thanabout 42.5° C., a formamide concentration of less than about 50%, and amoderate to low salt concentration. Presently preferred screeningconditions comprise a temperature of about 42.5° C., a formamideconcentration of about 20%, and a salt concentration of about 5×standardsaline citrate (SSC; 20×SSC contains 3M sodium chloride, 0.3M sodiumcitrate, pH 7.5). Such conditions will allow the identification ofsequences which have a substantial degree of similarity with the probesequence, without requiring perfect homology for the identification of astable hybrid. The phrase “substantial similarity” refers to sequenceswhich share at least 50% homology. Preferably, hybridization conditionswill be selected which allow the identification of sequences having atleast 70% homology with the probe, while discriminating againstsequences which have a lower degree of homology with the probe.

[0128] As used herein, a nucleic acid “probe” is single-stranded DNA orRNA, or analogs thereof, that has a sequence of nucleotides thatincludes at least 14, preferably at least 20, more preferably at least50, contiguous bases that are the same as (or the complement of) any 14or more contiguous bases set forth in any of SEQ ID NOs: 1, 3, 5, 7 or 9or the CRF-RA₁-encoding portion of clone hctCRFR. Preferred regions fromwhich to construct probes include 5′ and/or 3′ coding sequences,sequences predicted to encode transmembrane domains, sequences predictedto encode cytoplasmic loops, signal sequences, ligand binding sites, andthe like. The entire cDNA molecule encoding an invention CRF-R may alsobe employed as a probe. Probes may be labeled by methods well-known inthe art, as described hereinafter, and used in various diagnostic kits.

[0129] In accordance with yet another embodiment of the presentinvention, there is provided a method for the recombinant production ofinvention receptor(s) by expressing the above-described nucleic acidsequences in suitable host cells. The above-described nucleotidesequences can be incorporated into vectors for further manipulation. Asused herein, vector (or plasmid) refers to discrete elements that areused to introduce heterologous DNA (e.g., SEQ ID NOs:1, 3, 5, 7 or 9)into cells for either expression or replication thereof. Selection anduse of such vehicles are well within the skill of the artisan.

[0130] An expression vector includes elements capable of expressing DNAsthat are operatively linked with regulatory sequences (such as promoterregions) that are capable of regulating expression of such DNAfragments. Thus, an expression vector refers to a recombinant DNA or RNAconstruct, such as a plasmid, a phage, recombinant virus or other vectorthat, upon introduction into an appropriate host cell, results inexpression of the cloned DNA. Appropriate expression vectors are wellknown to those of skill in the art and include those that are replicablein eukaryotic cells and/or prokaryotic cells and those that remainepisomal or those which integrate into the host cell genome. Presentlypreferred plasmids for expression of invention CRF-Rs in eukaryotic hostcells, particularly mammalian cells, include cytomegalovirus (CMV)promoter-containing vectors, SV40 promoter-containing vectors, MMTV LTRpromoter-containing vectors, and the like.

[0131] As used herein, a promoter region refers to a segment of DNA thatcontrols transcription of DNA to which it is operatively linked. Thepromoter region includes specific sequences that are sufficient for RNApolymerase recognition, binding and transcription initiation. Thisportion of the promoter region is referred to as the promoter. Inaddition, the promoter region includes sequences that modulate thisrecognition, binding and transcription initiation activity of RNApolymerase. These sequences may be cis acting or may be responsive totrans acting factors. Promoters, depending upon the nature of theregulation, may be constitutive or regulated. Exemplary promoterscontemplated for use in the practice of the present invention includethe SV40 early promoter, the cytomegalovirus (CMV) promoter, the mousemammary tumor virus (MMTV) steroid-inducible promoter, Moloney murineleukemia virus (MMLV) promoter, and the like.

[0132] As used herein, the term “operatively linked” refers to thefunctional relationship of DNA with regulatory and effector sequences ofnucleotides, such as promoters, enhancers, transcriptional andtranslational stop sites, and other signal sequences. For example,operative linkage of DNA to a promoter refers to the physical andfunctional relationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA. In order to optimize expression and/or in vitro transcription, itmay be necessary to remove, add or alter 5′ untranslated portions of theclones to eliminate extra, potentially inappropriate alternativetranslation initiation (i.e., start) codons or other sequences that mayinterfere with or reduce expression, either at the level oftranscription or translation. Alternatively, consensus ribosome bindingsites (see, for example, Kozak (1991) J. Biol. Chem. 266:19867-19870)can be inserted immediately 5′ of the start codon and may enhanceexpression. The desirability of (or need for) such modification may beempirically determined.

[0133] As used herein, expression refers to the process by whichpolynucleic acids are transcribed into mRNA and translated intopeptides, polypeptides, or proteins. If the polynucleic acid is derivedfrom genomic DNA, expression may, if an appropriate eukaryotic host cellor organism is selected, include splicing of the mRNA.

[0134] Prokaryotic transformation vectors are well-known in the art andinclude pBlueskript and phage Lambda ZAP vectors (Stratagene, La Jolla,Calif.), and the like. Other suitable vectors for transformation of E.coli cells include the pET expression vectors (Novagen, see U.S. Pat.No. 4,952,496), e.g., pET11a, which contains the T7 promoter, T7terminator, the inducible E. coli lac operator, and the lac repressorgene; and pET 12a-c, which contain the T7 promoter, T7 terminator, andthe E. coli ompT secretion signal. Another suitable vector is thepIN-IIIompA2 (see Duffaud et al., Meth. in Enzymology, 153:492-507,1987), which contains the 1 pp promoter, the lacUV5 promoter operator,the ompA secretion signal, and the lac repressor gene.

[0135] Particularly preferred base vectors for transfection of mammaliancells are cytomegalovirus (CMV) promoter-based vectors such as pcDNA1(Invitrogen, San Diego, Calif.), MMTV promoter-based vectors such aspMAMNeo (Clontech, Palo Alto, Calif.) and pMSG (Catalog No. 27-4506-01from Pharmacia, Piscataway, N.J.), and SV40 promoter-based vectors suchas pSVb (Clontech, Palo Alto, Calif.), and the like.

[0136] The use of a wide variety of organisms has been described for therecombinant production of proteins or biologically active fragmentsthereof. One of skill in the art can readily determine suitable hosts(and expression conditions) for use in the recombinant production of thepeptides of the present invention. Yeast hosts, bacterial hosts,mammalian hosts, and the like can be employed.

[0137] In accordance with another embodiment of the present invention,there are provided “recombinant cells” containing the nucleic acidmolecules (i.e., DNA or mRNA) of the present invention (e.g., SEQ IDNOs:1, 3, 5, 7 or 9). Methods of transforming suitable host cells, aswell as methods applicable for culturing said cells containing a geneencoding a heterologous protein, are generally known in the art. See,for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA(1989).

[0138] Exemplary methods of transformation include, e.g., transformationemploying plasmids, viral, or bacterial phage vectors, transfection,electroporation, lipofection, and the like. The heterologous nucleicacid can optionally include sequences which allow for itsextrachromosomal maintenance, or said heterologous nucleic acid can becaused to integrate into the genome of the host (as an alternative meansto ensure stable maintenance in the host).

[0139] Host organisms contemplated for use in the practice of thepresent invention include those organisms in which recombinantproduction of heterologous proteins has been carried out. Examples ofsuch host organisms include bacteria (e.g., E. coli), yeast (e.g.,Saccharomyces cerevisiae, Candida tropicalis, Hansenula polymorpha andP. pastoris; see, e.g., U.S. Pat. Nos. 4,882,279, 4,837,148, 4,929,555and 4,855,231), mammalian cells (e.g., HEK293, CHO, CV-1, and Ltkcells), insect cells, and the like.

[0140] The present invention also provides a diagnostic system,preferably in kit form, for assaying for the presence of CRF-R protein,CRF-R polypeptide fragments or analogs, or CRF peptide in a fluid ortissue sample. A suitable diagnostic system includes, in an amountsufficient for at least one assay, a CRF-R protein (or polypeptidefragment thereof) and/or a subject antibody as a separately packagedimmunochemical reagent. Instructions for use of the packaged reagent arealso typically included.

[0141] “Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like.

[0142] In one embodiment, a diagnostic system for assaying for thepresence or quantity of CRF-R (or more likely a fragment of CRF-R) in avascular fluid sample, such as blood, plasma, or serum, or in a tissuesample, comprises a package containing at least one CRF-R protein orpolypeptide fragment thereof of this invention. In addition, adiagnostic system containing at least one CRF-R (or polypeptide fragmentthereof) can be used to detect the level of CRF peptide present in avascular fluid sample or to detect the presence of an intracellularG-protein.

[0143] In another embodiment, a diagnostic system of the presentinvention for assaying for the presence or amount of CRF-R or fragmentor analog thereof in a sample includes an anti-CRF-R antibodycomposition of this invention.

[0144] In yet another embodiment, a diagnostic system of the presentinvention for assaying for the presence or amount of CRF-R or a CRF-Rpolypeptide in a sample contains at least one CRF-R (or polypeptidefragment thereof) and an anti-CRF-R antibody composition of thisinvention.

[0145] In preferred embodiments, a diagnostic system of the presentinvention further includes a label or indicating means capable ofsignaling the formation of a complex containing a nucleic acid probe,protein, polypeptide, or antibody molecule of the present invention.

[0146] Also contemplated are immunohistochemistry diagnostic systems forcarrying out post-mortem diagnosis of mammalian tissue samples for thepresence of CRF-R, which employ the anti-CRF-R antibodies describedherein. For details on such diagnostic systems see, for example, Potteret al., PNAS, 89:4192-4296 (1992), incorporated herein by reference.

[0147] In yet another embodiment of the present invention, ahybridization histochemistry diagnostic system is provided. Thisdiagnostic system is useful for assaying for the presence or amount ofnucleic acid encoding CRF-R (e.g., CDNA or mRNA) in a sample (e.g.,vascular fluid or cellular tissue). This diagnostic system employs atleast one CRF-R encoding nucleic acid probe of this invention.

[0148] The word “complex” as used herein refers to the product of aspecific binding reaction such as an antibody:antigen, receptor:ligand,protein:protein, or nucleic-acid-probe:nucleic-acid-target reaction.Exemplary complexes are immunoreaction products and CRF:CRF-R complexes.

[0149] As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal. Any label or indicating means can be linked to or incorporatedin a nucleic acid probe, an expressed protein, polypeptide fragment, orantibody molecule that is part of an antibody or monoclonal antibodycomposition of the present invention, or used separately. These atoms ormolecules can be used alone or in conjunction with additional reagents.Such labels are themselves well-known in clinical diagnostic chemistry.

[0150] The labeling means can be a fluorescent labeling agent thatchemically binds to antibodies or antigens without denaturation to forma fluorochrome (dye) that is a useful immunofluorescent tracer. Suitablefluorescent labeling agents are fluorochromes such as fluoresceinisocyanate (FIC), fluorescein isothiocyanate (FITC),5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB-200-SC), and the like. A description ofimmunofluorescence analytic techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

[0151] In preferred embodiments, the indicating group is an enzyme, suchas horseradish peroxidase (HRP), glucose oxidase, and the like. In suchcases where the principal indicating group is an enzyme, additionalreagents are required for the production of a visible signal. Suchadditional reagents for HRP include hydrogen peroxide and an oxidationdye precursor such as diaminobenzidine. An additional reagent usefulwith glucose oxidase is 2,2′-azino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

[0152] In another embodiment, radioactive elements are employed aslabeling agents. An exemplary radiolabeling agent is a radioactiveelement that produces gamma ray emissions. Elements which emit gammarays, such as ¹²⁴I, ¹²⁵I, ¹²⁶I, ¹³¹I and ⁵¹Cr, represent one class ofradioactive element indicating groups. Particularly preferred is ¹²⁵I.Another group of useful labeling means are those elements such as ¹¹C,¹⁸F, ¹⁵O and ¹³N which emit positrons. The positrons so emitted producegamma rays upon encounters with electrons present in the animal's body.Also useful is a beta emitter, such as ³²P, ¹¹¹indium or ³H.

[0153] The linking of a label to a substrate, i.e., labeling of nucleicacid probes, antibodies, polypeptides, and proteins, is well known inthe art. For instance, antibody molecules can be labeled by metabolicincorporation of radiolabeled amino acids provided in the culturemedium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981).Conventional means of protein conjugation or coupling by activatedfunctional groups are particularly applicable. See, for example,Aurameas et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23 (1978),Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.

[0154] The diagnostic systems can also include, preferably as a separatepackage, a specific binding agent. A “specific binding agent” is amolecular entity capable of selectively binding a reagent species of thepresent invention or a complex containing such a species, but is notitself a polypeptide or antibody molecule composition of the presentinvention. Exemplary specific binding agents are second antibodymolecules (e.g., anti-Ig antibodies), complement proteins or fragmentsthereof, S. aureus protein A, and the like. Preferably the specificbinding agent binds the reagent species when that species is present aspart of a complex.

[0155] In preferred embodiments, the specific binding agent is labeled.However, when the diagnostic system includes a specific binding agentthat is not labeled, the agent is typically used as an amplifying meansor reagent. In these embodiments, the labeled specific binding agent iscapable of specifically binding the amplifying means when the amplifyingmeans is bound to a reagent species-containing complex.

[0156] The diagnostic kits can be used in an “ELISA” format to detectthe quantity of CRF, CRF-R, or CRF:CRF-R complex in a vascular fluidsample such as blood, serum, or plasma or in a mammalian tissue sample.“ELISA” refers to an enzyme-linked immunosorbent assay that employs anantibody or antigen bound to a solid phase and an enzyme-antigen orenzyme-antibody conjugate to detect and quantify the amount of anantigen present in a sample. A description of the ELISA technique isfound in Chapter 22 of the 4th Edition of Basic and Clinical Immunologyby D. P. Sites et al., published by Lange Medical Publications of LosAltos, Calif. in 1982 and in U.S. Pat. No. 3,654,090, U.S. Pat. No.3,850,752; and U.S. Pat. No. 4,016,043, which are all incorporatedherein by reference.

[0157] Thus, in preferred embodiments, CRF-R protein, a CRF-Rpolypeptide fragment thereof, a polyclonal anti-CRF-R antibody, or amonoclonal anti-CRF-R antibody is affixed to a solid matrix to form asolid support that comprises a package in the subject diagnosticsystems. A reagent is typically affixed to a solid matrix by adsorptionfrom aqueous medium, although other modes of affixation applicable toproteins and polypeptides well known to those skilled in the art can beused.

[0158] Useful solid matrices are also well known in the art. Suchmaterials are water insoluble and include cross-linked dextran(available from Pharmacia Fine Chemicals; Piscataway, N.J.); agarose;beads of polystyrene about 1 micron to about 5 millimeters in diameter(available from Abbott Laboratories; North Chicago, Ill.); polyvinylchloride; polystyrene; cross-linked polyacrylamide; nitrocellulose- ornylon-based webs such as sheets, strips or paddles; or tubes, plates orthe wells of a microtiter plate such as those made from polystyrene orpolyvinylchloride.

[0159] The reagent species, labeled specific binding agent or amplifyingreagent of any diagnostic system described herein can be provided insolution, as a liquid dispersion or as a substantially dry power, e.g.,in lyophilized form. Where the indicating means is an enzyme, theenzyme's substrate can also be provided in a separate package of asystem. A solid support such as the before-described microtiter plateand one or more buffers can also be included as separately packagedelements in this diagnostic assay system.

[0160] The packaging materials contemplated herein in relation todiagnostic systems are those customarily utilized in diagnostic systems.The term “package” refers to a solid matrix or material such as glass,plastic (e.g., polyethylene, polypropylene and polycarbonate), paper,foil, and the like, capable of holding within fixed limits a diagnosticreagent such as a protein, polypeptide fragment, antibody or monoclonalantibody of the present invention. Thus, for example, a package can be abottle, vial, plastic or plastic-foil laminated envelope container, orthe like, used to contain a diagnostic reagent. Alternatively, thecontainer used can be a microtiter plate well to which microgramquantities of a diagnostic reagent have been operatively affixed, i.e.,linked so as to be capable of being immunologically bound by an antibodyor polypeptide to be detected.

[0161] In normal individuals, the levels of CRF can vary from about 1 to28 picograms per milliliter of vascular fluid. However, during the lasttrimester of pregnancy, it has been found that there is a tendency forCRF levels to prematurely increase. It is believed that this increase isassociated with pregnancy-induced hypertension. Monitoring the change inthe level of CRF could facilitate the prediction of the possibility ofpremature labor, which can be avoided by appropriate treatment.

[0162] Thus, by monitoring the level of CRF, an abnormal increaseindicative of a potential pathological problem in pregnancy can bedetected at an early stage. Because normal hypertension is now believedto be either caused (or accompanied by) a higher CRF/“CRF-bindingprotein” ratio than normal, monitoring the level of CRF facilitates theprediction of particular patients who are predisposed to such diseases,and permits therapeutic intervention—as for example by administeringdosages of CRF-R protein or polypeptide fragments thereof. By theadministration of CRF-R or fragments thereof to treat such pregnancyrelated disorders, CRF levels can be returned to normal, thusfacilitating the normal growth of the fetus.

[0163] The present invention contemplates various immunoassay methodsfor determining the amount of CRF-R in a biological fluid or tissuesample using a CRF-R, a polypeptide fragment thereof (includingimmunologic fragments, i.e., fragments capable of generating an immuneresponse, as more fully described in Harlowe and Lane, Antibodies: ALaboratory Manual, p. 76 (Cold Spring Harbor Laboratory, 1988)), ananti-CRF-R polyclonal or monoclonal antibody of this invention as animmunochemical reagent to form an immunoreaction product whose amountrelates, either directly or indirectly, to the amount of CRF-R in thesample. Also contemplated are immunoassay methods for determining theamount of CRF peptide in a biological fluid sample using a CRF-R or apolypeptide fragment thereof as a reagent to form a product whose amountrelates, either directly or indirectly, to the amount of CRF in thesample.

[0164] Various well-known heterogenous and homogenous protocols, eithercompetitive or noncompetitive, solution-phase or solid-phase, can beemployed in performing assay methods of the present invention. Thoseskilled in the art will understand that there are numerous well knownclinical diagnostic chemistry procedures in which an immunochemicalreagent of this invention can be used to form an immunoreaction productwhose amount relates to the amount of CRF-R or CRF present in a bodysample.

[0165] In one embodiment, the detection of CRF-R protein or polypeptidefragments in a body sample is utilized as a means to monitor the fate oftherapeutically administered CRF-R or polypeptide fragments according tothe therapeutic methods disclosed herein.

[0166] Also contemplated are immunological assays capable of detectingthe formation of immunoreaction product formation without the use of alabel. Such methods employ a “detection means”, which means arethemselves well-known in clinical diagnostic chemistry. Exemplarydetection means include biosensing methods based on detecting changes inthe reflectivity of a surface, changes in the absorption of anevanescent wave by optical fibers, changes in the propagation of surfaceacoustical waves, and the like.

[0167] The present invention contemplates therapeutic compositionsuseful for practicing the therapeutic methods described herein.Therapeutic compositions of the present invention contain aphysiologically compatible carrier together with a CRF-R protein, CRF-Rpolypeptide fragment, or anti-CRF-R antibody, as described herein,dissolved or dispersed therein as an active ingredient. In a preferredembodiment, the therapeutic composition is not immunogenic whenadministered to a mammal or human patient for therapeutic purposes.

[0168] As used herein, the terms “pharmaceutically acceptable”,“physiologically compatible” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset, and thelike.

[0169] The preparation of a pharmacological composition that containsactive ingredients dissolved or dispersed therein is well known in theart. Typically such compositions are prepared as injectables either asliquid solutions or suspensions; however, solid forms suitable forsolution, or suspension, in liquid prior to use can also be prepared.The preparation can also be emulsified.

[0170] The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient inamounts suitable for use in the therapeutic methods described herein.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like, as well as combinations of any two or morethereof. In addition, if desired, the composition can contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, and the like, which enhance the effectiveness ofthe active ingredient.

[0171] The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable nontoxic salts include the acid additionsalts (formed with the free amino groups of the polypeptide) that areformed with inorganic acids such as, for example, hydrochloric acid,hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolicacid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinicacid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid,naphthalene sulfonic acid, sulfanilic acid, and the like.

[0172] Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and the like; and organic bases such asmono-, di-, and tri-alkyl and -aryl amines (e.g., triethylamine,diisopropyl amine, methyl amine, dimethyl amine, and the like) andoptionally substituted ethanolamines (e.g., ethanolamine,diethanolamine, and the like).

[0173] Physiologically tolerable carriers are well known in the art.Exemplary liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, polyethylene glycoland other solutes.

[0174] Liquid compositions can also contain liquid phases in addition toand to the exclusion of water. Exemplary additional liquid phasesinclude glycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

[0175] As previously indicated, administration of the CRF-Rs orpolypeptide fragments thereof is effective to reduce vascular fluid CRFlevels or high ACTH levels in mammals caused by excessive CRF, which isreferred to herein as “CRF-induced ACTH release.” In this manner, theCRF-Rs are useful in treating high cortisol (i.e., glucocorticoids)levels which are associated with hypercortisolemia, Cushing's Disease,alcoholism, anorexia nervosa and similar diseases. Likewise, theseCRF-Rs are considered to have utility in combatting pituitary tumorsthat produce CRF—particularly in maintaining stability in a patientuntil such a tumor can be surgically removed.

[0176] In accordance with the present invention, CRF-RB₁ hassurprisingly been found to be abundantly expressed in the heart. In theisolated perfused heart, the addition of CRF into the left atriuminduces a prolonged dilatory effect on coronary arteries, transientlyproduces a positive ionotropic effect, and stimulates the secretion ofatrial natriuretic peptide (Saitoh et al., Gen. Pharmac. (England)21:337-342 (1990); and Grunt et al., Horm. Metab. Res. (Germany)24:56-59 (1992)). The surprising finding of CRF-RB₁ expression in theblood vessels of the heart raises the possibility that CRF (or othernatural or pharmacologic ligands for this receptor) might regulatecardiac perfusion. Furthermore, it is expected that other vascular beds,such as the superior mesenteric, known to be dilated by CRF and relatedligands, will also be found to be regulated by CRF and receptorstherefor.

[0177] Accordingly, since it is known that CRF has a number ofbiological effects in the heart, it is contemplated that the CRF-Rproteins, fragments thereof, or agonists/antagonists thereof (e.g.,anti-CRF-R antibodies), can be effectively used to selectively modulatethe action of CRF on the heart, particularly in methods to increase thelevel of CRF that can act on the heart blood vessels to maintainpatency.

[0178] In accordance with the present invention, the CRF-RB gene hasalso been found to be expressed in the gastrointestinal tract, as shown,for example, by the presence of CRF-RB₁ mRNA in the submucosal anddeeper regions of the duodenum. Accordingly, the CRF-RB₁ receptor maymediate some of the direct stimulatory effects of CRF on the GI tractthat have been described. For example, CRF acts on the gut in vitro todepolarize myenteric neurons in the small intestine (Hanani and Wood,Eur. J. Pharmacol. (Netherlands) 211:23-27 (1992)). In vivo studies withintravenously administered CRF and CRF antagonists are consistent with adirect effect of CRF to control gastric emptying and intestinal motility(Williams et al., Am. J. Physiol. 253:G582-G586 (1987); Lenz, H. J.,Horm. Metab. Res. Suppl. 16:17-23 (1987); and Sheldon et al., Regul.Pept. 28:137-151 (1990)). In addition, CRF immunostaining is present atmany levels of the GI tract (Nieuwenhuyzen-Kruseman et al., Lancet2:1245-1246 (1982); Petrusz et al., Federation Proc. 44:229-235 (1985);and Kawahito et al., Gastroenterology 106:859-865 (1994)).

[0179] Thus, CRF-Rs (e.g., CRF-RB), fragments thereof, oragonists/antagonists thereof (e.g., anti-CRF-R antibodies), arecontemplated for use in treating gastrointestinal disorders, such asirritable bowel syndrome. In addition, CRF antagonists that areselective for CRF-RB are also contemplated for usef in therapeuticmethods to treat irritable bowel syndrome.

[0180] In addition, the presence of CRF-RB in the epididymis may enablelocal communication with spermatozoa, which are reported to possessimmunoreactive CRF (Yoon et a., Endocrinology 122:759-761 (1988)). Thus,CRF-Rs are also contemplated for use in treating fertility disorders.

[0181] The CRF-R proteins and fragments thereof are also useful to treatabnormalities, such as, for example, preeclampsia (toxemia ofpregnancy), which occur during pregnancy; for example, they can be usedto reduce pregnancy-induced complications and increased CRF levels whichcan otherwise result in excessive release of ACTH. In addition, CRF-Rproteins or fragments thereof can be administered to sequester CRF fromvascular fluid, thereby reducing the ratio of CRF/“CRF-binding protein”present in a patient, wherein it is beneficial to reduce the levels offree CRF (i.e., CRF not bound to CRF-BP) in the vascular fluid sample.CRF-binding protein (CRF-BP) is an extracellular serum protein describedin Potter et al., supra. The IV administration of CRF-Rs may also beemployed in certain instances to modulate blood pressure and therebycombat hypotension.

[0182] Since CRF is a known modulator of the immune system, it iscontemplated that the administration of CRF-R protein or fragmentsthereof may be useful to locally treat, i.e., by direct injection intothe affected joint, arthritis and other similar ailments. CRF is knownto have a number of biological effects on the pituitary, andaccordingly, the CRF-R proteins can be used to modulate the action ofCRF on the pituitary. Furthermore, it is well known that CRF has anumber of biological effects in the brain; therefore, it is contemplatedthat the CRF-R proteins can be effectively used to modulate the actionof CRF on the brain, particularly with respect to control of appetite,reproduction, growth, anxiety, depression, fever and metabolism, as wellas the regulation of blood pressure, heart rate, blood flow, and thelike.

[0183] Thus, the present invention provides for a method for modulatingthe action of CRF in mammals comprising administering a therapeuticallyeffective amount of a physiologically acceptable composition containingCRF-R protein or polypeptide fragment of the present invention. Inaddition, the stimulation of ACTH release by CRF can be enhanced bytransfecting the subject with a tissue specific CRF-encoding construct.

[0184] In another embodiment, the present invention provides a methodfor treating a pregnancy-related pathological disorder in mammalscomprising administering a therapeutically effective amount of aphysiologically acceptable composition containing a CRF-R protein orpolypeptide fragment of the present invention, said amount beingeffective to sequester CRF, thereby producing a CRF/“CRF-bindingprotein” ratio within the normal range for a pregnant female.

[0185] Also, as earlier indicated, the administration of anti-CRF-Rantibodies described herein is effective to modulate the biologicaleffect of CRF-Rs when administered in vivo. For example, an anti-CRF-Rantibody of this invention can be used in the above-described mammaliantherapeutic methods to: neutralize or counteract the effect of CRF-R,increase the level of free CRF (e.g., CRF not bound by CRF-R), decreaseCRF-induced ACTH release, or decrease the level of ACTH-inducedglucocorticoids in a subject. Because increasing the level of free CRFincreases the level of CRF-induced ACTH release, which increasesglucocorticoid production, these therapeutic methods are useful fortreating certain physiological conditions where increasing the level ofglucocorticoids in a patient's vascular fluid is therapeuticallyeffective, such as conditions of inflammation or Addison's Disease, andthe like.

[0186] Administration of antibodies for this purpose would be carriedout along the lines and in amounts generally known in this art, and moreparticularly along the lines indicated herein with respect toadministration of the protein itself.

[0187] As described herein, a therapeutically effective amount is apredetermined amount calculated to achieve the desired effect, e.g., todecrease the amount of CRF, ACTH, or decrease the CRF/“CRF-bindingprotein” ratio in a patient. The required dosage will vary with theparticular treatment and with the duration of desired treatment;however, it is anticipated that dosages between about 10 micrograms andabout 1 milligram per kilogram of body weight per day will be used fortherapeutic treatment. It may be particularly advantageous to administersuch compounds in depot or long-lasting form as discussed hereinafter. Atherapeutically effective amount is typically an amount of a CRF-Rprotein or polypeptide fragment thereof that, when administered in aphysiologically acceptable composition, is sufficient to achieve aplasma concentration of from about 0.1 mg/ml to about 100 mg/ml,preferably from about 1.0 mg/ml to about 50 mg/ml, more preferably atleast about 2 mg/ml and usually 5 to 10 mg/ml. Antibodies areadministered in proportionately appropriate amounts in accordance withknown practices in this art.

[0188] The level of ACTH present in a patient, particularly in theplasma, can be readily determined by routine clinical analysis. Inaddition, changes in ACTH levels can be monitored during a treatmentregimen to determine the effectiveness of the administered CRF-R proteinor polypeptide fragment over time.

[0189] Thus, the present therapeutic method provides an in vivo meansfor decreasing ACTH levels in a subject displaying symptoms of elevatedserum ACTH, or is otherwise at medical risk by the presence of serumACTH, wherein it is beneficial to reduce the levels of ACTH. Inaddition, the present therapeutic method provides an in vivo means fordecreasing ACTH-induced cortisol levels (e.g., glucocorticoids) in ahuman patient displaying symptoms of elevated serum cortisol.

[0190] Likewise, the level of CRF present in a patient, particularly inthe plasma, can be readily determined by the diagnostic methods and kitsprovided herein and readily manipulated by administering CRF-R, analogsthereof, or anti-CRF-R antibodies.

[0191] Thus, the present therapeutic method provides an in vivo meansfor decreasing the CRF/CRF-BP ratio in a subject displaying symptoms ofelevated serum CRF/CRF-BP levels, or is otherwise at medical risk by thepresence of an elevated serum CRF/CRF-BP ratio, wherein it is beneficialto reduce the levels of free CRF (i.e., CRF not bound to CRF-BP) in thevascular fluid sample.

[0192] CRF-R protein(s) (or functional fragments thereof) should beadministered under the guidance of a physician. Pharmaceuticalcompositions will usually contain the protein in conjunction with aconventional, pharmaceutically-acceptable carrier. For treatment,substantially pure synthetic CRF-R or a nontoxic salt thereof, combinedwith a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition, is preferably administered parenterally to mammals,including humans, either intravenously, subcutaneously, intramuscularly,percutaneously, e.g. intranasally, or intracerebroventricularly; oraladministration is possible with an appropriate carrier.

[0193] Therapeutic compositions containing CRF-R polypeptide(s) of thisinvention are preferably administered intravenously, as by injection ofa unit dose, for example. The term “unit dose,” when used in referenceto a therapeutic composition of the present invention, refers tophysically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent, i.e., carrier, or vehicle.

[0194] Compositions are administered in a manner compatible with thedosage formulation, and in a therapeutically effective amount. Thequantity to be administered depends on the subject to be treated,capacity of the subject's immune system to utilize the activeingredient, and degree of therapeutic effect desired. Precise amounts ofactive ingredient required to be administered depend on the judgment ofthe practitioner and are peculiar to each individual. However, suitabledosage ranges for systemic application are disclosed herein and dependon the route of administration. Suitable regimes for initialadministration and booster shots are also variable, but are typified byan initial administration followed by repeated doses at one or moreintervals by a subsequent injection or other administration.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations in the blood in the ranges specified for in vivotherapies are contemplated.

[0195] As an aid to the administration of effective amounts of a CRF-Rpolypeptide, a diagnostic method of this invention for detecting a CRF-Rpolypeptide in the subject's blood is useful to characterize the fate ofthe administered therapeutic composition.

[0196] It may also be desirable to deliver CRF-R over prolonged periodsof time, for example, for periods of one week to one year from a singleadministration, and slow release, depot or implant dosage forms may beutilized. For example, a dosage form may contain a pharmaceuticallyacceptable non-toxic salt of the compound which has a low degree ofsolubility in body fluids, for example, an acid addition salt with thepolybasic acid; a salt with a polyvalent metal cation; or combination ofthe two salts. A relatively insoluble salt may also be formulated in agel, for example, an aluminum stearate gel. A suitable slow releasedepot formulation for injection may also contain CRF-R or a salt thereofdispersed or encapsulated in a slow degrading, non-toxic ornon-antigenic polymer such as a polylactic acid/polyglycolic acidpolymer, for example, as described in U.S. Pat. No. 3,773,919. Thesecompounds may also be formulated into silastic implants.

[0197] As additional examples of the utility of invention compositions,nucleic acids, receptors and/or antibodies of the invention can be usedin such areas as the diagnosis and/or treatment of CRF-dependent tumors,enhancing the survival of brain neurons, inducing abortion in livestockand other domesticated animals, inducing twinning in livestock and otherdomesticated animals, and so on.

[0198] In addition, invention cDNAs described herein encoding CRF-Rs(e.g., CRF-RA and CRF-RB) can be used to isolate genomic clones encodingthe respective CRF-R gene. The 5′ regulatory region of the isolatedCRF-R gene can be sequenced to identify tissue-specific transcriptionelements (i.e., promoter). The tissue-specific CRF-R promoters obtainedare useful to target various genes to cells that normally expressCRF-Rs. For example, an adenovirus vector, having DNA encoding acytotoxic protein and a tissue-specific CRF-R promoter for pituitarycorticotropic cells, can be used as means for killing pituitarycorticotropic tumor cells.

[0199] The invention will now be described in greater detail byreference to the following non-limiting examples.

EXAMPLES

[0200] Unless otherwise stated, the present invention was performedusing standard procedures, as described, for example in Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., MolecularCloning: A Laboratory Manual (2 ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methodsin Molecular Biology, Elsevier Science Publishing, Inc., New York, USA(1986); Methods in Enzymology: Guide to Molecular Cloning TechniquesVol.152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., SanDiego, USA (1987); or Harlowe and Lane, Antibodies: A Laboratory Manual,p. 76 (Cold Spring Harbor Laboratory, 1988).

[0201] Double-stranded DNA was sequenced by the dideoxy chaintermination method using the Sequenase reagents from US Biochemicals.Comparison of DNA sequences to databases was performed using the FASTAprogram [Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444-2448(1988)]. Tyr-ovine CRF used for iodination was purchased from Peninsula.

Example 1 Isolation of cDNA Encoding a Human CRF-R

[0202] A cDNA library of approximately 1.5×10⁶ independent clones fromhuman pituitary corticotrope adenoma (Cushing's Tumor) cells wasconstructed in the mammalian expression vector, pcDNA1, and screenedusing an expression cloning approach [Gearing et al., EMBO J. 8,3667-3676 (1989)] based on the ability of single transfected cells todetectably bind labeled ¹²⁵I-Tyr-ovine CRF. Binding was assessed byperforming the transfections and binding reactions directly on chamberedmicroscope slides, then dipping the slides in photographic emulsion,developing the slides after 3-4 days exposure, and analyzing them undera microscope. The possibility of detecting expressed CRF BindingProtein, CRF-BP, rather than authentic CRF-R, was minimized by theselection of an ovine CRF related tracer known to have high affinity forthe receptor but low affinity for CRF-BP. Cells which had beentransfected with CRF receptor cDNA, and consequently bound radioactiveCRF, were covered with silver grains.

[0203] Polyadenylated RNA was prepared from human pituitary corticotropeadenoma cells. Corresponding cDNA was synthesized and ligated into theplasmid vector pcDNA1 using non-palindromic BstXI linkers and used totransform MC1061/P3 cells, yielding a library of approximately 1.5×10⁶primary recombinants. The unamplified cDNA library was plated atapproximately 5000 clones per 100 mm plate. The cells were then scrapedoff the plates, frozen in glycerol, and stored at −70° C.

[0204] Mini-prep DNA was prepared from each pool of 5,000 clones usingthe alkaline lysis method [Maniatis et al. Molecular Cloning (ColdSpring Harbor Laboratory (1982)]. Approximately {fraction (1/10)} of theDNA from a mini-prep (10 ml of 100 ml) was transfected into COSM6 cells,and the cells screened for the capacity to bind iodinated Tyr ovine CRF.

[0205] More specifically, 2×10⁵ COS cells were plated on chamberedmicroscope slides (1 chamber-Nunc) that had been coated with 20 mg/mlpoly-D-lysine and allowed to attach for at least 3 hours in DMEM and10Fetal Calf Serum (complete medium). Cells were subjected toDEAE-Dextran mediated transfection as follows. 1.5 ml of serum-freeDulbecco's Modified Eagle's medium (DMEM) containing 100 mM chloroquinewas added to the cells. DNA was precipitated in 200 ml DMEM/chloroquinecontaining 500 mg/ml DEAE-Dextran, then added to the cells. The cellswere incubated at 37° C. for 4 hours, then the media was removed and thecells were treated with 10% DMSO in HEPES buffered saline for 2 minutes.The 10% DMSO was removed, and fresh complete media was added and thecells assayed for binding 2 days later.

[0206] Transfected cells prepared as described above were washed twicewith HEPES buffered saline (HDB) containing 0.1% ovalbumin, thenincubated for 90 minutes at 22° C. in 0.7 ml HDB, 0.1% ovalbumincontaining 10⁶ cpm ¹²⁵I-Tyr-ovine CRF (approximately 1 ng, 300 pM). Thecells were then washed 3× with cold HDB, 0.1% ovalbumin, and 2× withcold HDB, then fixed for 15 minutes at 22° C. in 2.5% glutaraldehyde/HDBand washed 2× with HDB. The chambers were then peeled off the slides,and the slides dehydrated in 95% ethanol, dried under vacuum, dipped inNTB2 photographic emulsion (Kodak) and exposed in the dark at 4° C. for3-4 days. Following development of the emulsion, the slides weredehydrated in 95% ethanol, stained with eosin and coverslipped with DPXmountant (Electron Microscopy Sciences). The slides were analyzed underdarkfield illumination using a Leitz microscope.

[0207] Successive subdivision of a positive pool generated a singleclone that demonstrated high affinity CRF binding (K_(d)=3.3±0.45 nM)when present in COSM6 cell membranes. The clone containing sequenceencoding CRF-receptor is referred to herein as “hctCRFR” and has beendeposited with ATCC under accession number 75474, and the receptorencoded thereby is referred to herein as hCRF-RA₁.

[0208] A phage 1 ZapII library was also synthesized from the same humanCushing's tumor cDNA described above using NotI/EcoRI adapters. A 1.2 kbPstI fragment in the CRF-R coding region of clone “hctCRFR” was used toscreen the 1 ZapII library at high stringency using standard methods. Ofthree positive clones identified, two were sequenced and found tocontain full length CRF-R cDNA without introns. The clones are labeled“CRF-R1” (also referred to herein as hCRF-RA₁) and “CRF-R2” (alsoreferred to herein as hCRF-RA₂), portions of which are set forth in SEQID NO:1 and SEQ ID NO:3, respectively. Clone CRF-R₁ (i.e., hCRF-RA₁)contains a 2584 bp insert with a 1245 bp open reading frame encoding a415 amino acid CRF-R protein. Clone CRF-R2 (also referred to herein ashCRF-RA₂), is an alternatively spliced variant sequence of CRF-R1 (i.e.,hCRF-RA₁) that has the 29 amino acids set forth in SEQ ID NO:4 insertedbetween amino acids 145-146 of SEQ ID NO:2.

Example 2 Expression of CRF Receptor mRNA

[0209] Using well-known autoradiographic methods for binding labelledCRF to various frozen tissue sections, the native CRF receptor has beendetected and shown to vary dynamically in the pituitary and variousbrain regions in experimental animals and in human beings where it isaltered in pathologic conditions including Alzheimer's Disease andsevere melancholic depression. Furthermore, receptors have been detectedin the periphery in organs such as the adrenal, ovary, placenta,gastrointestinal tract and the red pulp, macrophage rich area of thespleen and in sites of inflammation presumably corresponding to theactions of CRF within those tissues.

[0210] A Northern-blot assay was conducted by size-fractionatingpoly(A)⁺-RNA (derived from rat brain, rat pituitary, rat heart, andmouse AtT20 corticotropic cells) on a denaturing formaldehyde agarosegel and transferring the RNA to nitrocellulose paper using standardmethods. The nitrocellulose paper blot was prehybridized for 15 minutesat 68° C. in QuikHybÔ hybridization solution (Stratagene, La Jolla,Calif.) and 100 mg/ml salmon sperm DNA. Next, the blot was hybridized inthe same solution at 68° C. for 30 minutes to a “hctCRFR”-derivedrandomly primed (Amersham, Arlington Heights, Ill.) 1.3 Kb PstI cDNAfragment that contained the majority of the cDNA region of CRF-R1 (i.e.,hCRF-RA₁). The blot was washed twice at 21° C. in 2×SSPE and 0.15%Sodium Dodecyl Sulfate (SDS) for 15 minutes. Next, the blot was washedtwice at 60° C. in 0.2×SSPE and 0.1% SDS for 30 minutes. Anautoradiogram of the nitrocellulose paper blot was developed usingstandard methods.

[0211] The results of the Northern-blot assay revealed the presence of a2.7 Kb CRF-R mRNA transcript in rat brain, rat pituitary, and in mouseAtT20 corticotropic cells. CRF-R mRNA was not detected in the hearttissue sample.

Example 3 Pharmacologic Characteristics of hctCRFR Transiently Expressedin COSM6 Cells

[0212] Approximately 10⁶ COSM6 cells were transfected with eitherhctCRFR or rGnRHR (rat gonadotropin releasing hormone receptor) by theDEAE-dextran method and grown in 150 mm tissue culture dishes. Two daysafter transfection, the cells were washed twice with 1 ml HDB and weredetached by incubation for 15 min at room temperature in 0.5 mM EDTA inHDB. After pelleting, the cells were washed twice with HDB, and thenhomogenized in 5% sucrose (16 ml/150 mm dish). The homogenate wascentrifuged at 600×g for 5 minutes, and the resulting supernatant wascentrifuged at 40,000×g for 20 minutes. The resulting pellet (containingcrude membranes) was resuspended at 1-4 mg/ml in 10% sucrose, and usedin a competitive radioreceptor assay to measure binding to the CRF-R asdescribed in Perrin et al., Endoc., 118:1171 (1986).

[0213] Membrane homogenates (10-24 mg) were incubated at roomtemperature for 90 minutes with 100,000 cpm ¹²⁵I-(Nle²¹, Tyr³²)-ovineCRF (1 mg CRF was iodinated by chloramine T oxidation to a specificactivity of 2,000 Ci/mmol; iodinated CRF was purified by HPLC) andincreasing concentrations of unlabeled rat/human (r/h) CRF. Theiodinated CRF and unlabeled r/h CRF were both diluted in 20 mM HEPES,0.1% BSA, 10% sucrose, 2 mM EGTA to a final, pH 7.5 in a final volume of200 ml and containing MgSO₄ to a final concentration of 10 mM. Thereaction was terminated by filtration through GF/C (Whatman) filters,prewetted with 1% BSA, 10 mM HEPES, pH 7.5. The filters were washed 4times with 1 ml 0.1% BSA, 50 mM Tris, pH 7.5. Filter-boundradioactivity, indicating the presence of CRF-R: ¹²⁵I-(Nle²¹,Tyr³²)-ovine CRF complex, was determined by g-scintillation counting.

[0214] The results from an assay for the displacement of ¹²⁵I-(Nle²¹,Tyr³²)-ovine CRF by unlabeled human/rat CRF (r/h CRF) are shown inFIG. 1. The results show that native r/h CRF is able to displace labeledovine CRF in a dose-dependent manner from cells transfected withhctCRFR, but not from cells transfected with rGnRHR. This indicates thatthe hctCRFR clone encodes a receptor that displays pharmacologicspecificity characteristic of a physiologically relevant CRF-receptor(i.e., CRF-RA₁).

Example 4 Assay of CRF-R Mediated Stimulation of Intracellular cAMPLevels

[0215] To determine the possible linkage of CRF-R to multiple signalingpathways, the ability of CRF-R to stimulate cAMP formation inCRF-R-expressing COSM6 cells was investigated. To ensure that changes incAMP levels were not influenced by alterations in cAMPphosphodiesterase, the phosphodiesterase inhibitor3-isobutyl-1-methylxanthine (IBMX) was added to the medium. COSM6 cellswere trypsinized 24 hrs following transfection with either ctCRFR orrGnRHR in 150 mm dishes and were replated in 24-well plates (Costar) andallowed to express the receptors for another 24 hrs in 10% FCS, DMEM.

[0216] On the day of the stimulation, the medium was changed to 0.1 FCS,DMEM at least 2 hrs before a 30 minute preincubation with 0.1 mM IBMX ormedium. Test ligands (i.e., r/h CRF, sauvagine, salmon calcitonin,Vasoactive intestinal peptide (VIP), growth hormone releasing factor(GRF) were added in 0.1% BSA, 0.1% FCS, DMEM, and stimulation wascarried out for 30 minutes at 37° C., 7.5% CO₂. The medium was removedand the cells were extracted overnight with 1 ml ice-cold 95% EtOH-0.1MHCl at −20° C. Cyclic AMP (cAMP) levels were determined in duplicatefrom triplicate wells by RIA kit (Biomedical Technologies, Stoughton,Mass.) following the manufacturer's protocol.

[0217] The results are shown in FIGS. 2A, 2B and 2C. FIGS. 2A and 2Bshow that COSM6 cells transfected with the cloned hctCRFR respond to CRFwith an approximately 10-20 fold increase in intracellular cAMP overbasal cAMP levels. Several unrelated peptides have no effect on cyclicAMP levels in the receptor transfected cells. FIG. 2C shows that the CRFantagonist, a helical (9-41) CRF, blocks the induction of cyclic AMP byr/h CRF.

Example 5 Isolation of cDNA Encoding a Rat CRF-R

[0218] Adult Sprague-Dawley rat whole brain poly (A)+RNA was used forthe synthesis of a cDNA library. Double-stranded cDNA was ligated toEcoRI-NotI adaptors (Pharmacia/LKB) and cDNAs greater than 2 kilobasepair (kb) were ligated into the 1 ZAPII vector (Stratagene, La Jolla,Calif.). The library was amplified once and approximately 7×10⁵ cloneswere screened by hybridization with the 1.2 kb PstI fragment of CRF-R1(e.g., CRF-RA₁) using standard methods. One of the positive clonesidentified was sequenced and found to contain full length CRF-R cDNA.The positive clone was labeled rat brain CRF-R (rbCRF-RA) and containsan approximate 2500 base pair (bp) insert with a 1245 bp open readingframe encoding a 415 amino acid CRF-R protein. The cDNA and amino acidsequences corresponding to “rbCRF-RA” are set forth in SEQ ID NOs: 5 and6, respectively.

Example 6 Isolation of Genomic DNA Encoding a Mouse CRF-RB₁

[0219] Approximately 7×10⁶ clones of a mouse phage genomic library(obtained from Stratagene, La Jolla, Calif.) were screened byhybridization with a probe comprising nucleotides 204-1402 of rat CRF-RA(see SEQ ID NO:5) using standard methods. Thus, hybridization wascarried out in 5×SSPE, 5×Denhardt's solution, and 0.5% SDS for 16 hoursat 60° C. The filters were washed twice at room temperature with 2×SSC,0.1% SDS, then washed twice at 60° C. with 2×SSC, 0.1% SDS.

[0220] One of the positive clones identified was sequenced and found tocontain an open reading frame encoding a partial CRF-RB₁ sequencederived from transmembrane domains 3 through 4 of CRF-RB₁. The positiveclone was labeled mouse CRF-RB₁ (mCRF-RB₁) and contains two exons,interrupted by an intron of about 450 nucleotides. The two exons combineto produce a 210 base pair (bp) open reading frame encoding a 70 aminoacid portion of a novel CRF-RB₁ protein. The cDNA and amino acidsequences corresponding to “mCRF-RB₁” are set forth in SEQ ID NOs: 7 and8, respectively.

[0221] Upon further sequencing of clone mCRF-RB₁, a third exon wasrevealed containing an additional 78 base pairs (corresponding tonucleotides 895-972 of SEQ ID NO:9) of an open reading frame encodingthe CRF-RB₁ protein.

Example 7 Isolation of cDNA Encoding a Mouse CRF-RB₁

[0222] In order to obtain the cDNA corresponding to the new mouseCRF-RB₁ receptor, a mouse heart phage library was screened byhybridization. Approximately 1.2×10⁶ phage plaques of an amplifiedoligo-dt primed mouse heart cDNA library in the lambda zap II vector(Stratagene) were screened by hybridization. A probe for the CRF-RB₁cDNA was prepared by PCR using [a³²P]-dCTP and the following primers:

[0223] sense: 5′ CTGCATCACCACCATCTTCAACT 3′ (SEQ ID NO:11); and

[0224] antisense: 5′ AGCCACTTGCGCAGGTGCTC 3′ (SEQ ID NO:12).

[0225] The template used in generating the probe was plasmid DNAcorresponding to one exon of CRF-RB₁ extending from amino acids 206 to246 of SEQ ID NO:10. PCR amplification was carried out for 30 cycles(denatured at 94° C. for 1 minute, annealed at 55° C. for 2 minutes andextended at 72° C. for 3 minutes) to yield a 123 bp productcorresponding to nucleotides 693-815 of SEQ ID NO:9.

[0226] For hybridization screening, the plaques were lifted onto nylonmembranes, then denatured, neutralized and rinsed. The membranes wereprehyrbridized at 42° C. in 0.6M NaCl, 60 mM sodium citrate,4×Denhardt's, 40 mM sodium phosphate, pH 6.5, 170 mg/ml salmon spermDNA, 0.1% SDS, 20% formamide. The membranes were then hybridized at 42°C. in the same solution plus 50% dextran sulfate with approximately 10⁶cpm of labeled probe per ml of solution. After hybridization, thefilters were washed with 0.3M NaCl, 30 mM sodium citrate, 0.1% SDS onceat room temperature, twice at 42° C., and twice at 50° C.

[0227] A positive plaque was isolated and purified in the next round ofplaque hybridization. Helper phage R408 (Biorad) was used for in vivoexcision of the lambda zap II clone. The cloned receptor was sequencedon both strands by the dideoxy chain-termination method using theSequenase kit (United States Biochemical). The clone encoding mouseCRF-RB₁ contained a 2.2 kb insert that included a 1293 base pair (bp)open reading frame encoding a protein of 431 amino acids. Thefull-length CRF-RB₁ receptor cDNA was subcloned into the expressionvector pcDNA1 (Invitrogen) using the EcoRI restriction enzyme to producethe plasmid pCRF-RB₁.

[0228] Alignments of the nucleotide and amino acid sequences werecarried out using the Jotun-Hein weighted method and the PAM250 residueweight table, respectively. CRF-RB₁ has 70% homology at the nucleotidelevel and 68% homology at the amino acid level, to CRF-RA₁. FIGS. 3A and3B present the comparison between the amino acid sequences of CRF-RB₁and CRF-RA₁. The alignment was selected to maximize the regions ofsimilarity. There is a putative signal peptide and five putativeN-glycosylation sites in the N-terminal domain in CRF-RB₁, as there arein CRF-RA₁. When comparing CRF-RB₁ to CRF-RA₁, there is 79% similarityin the seven transmembrane domains (TMD) and 84% similarity in theintracellular loops and the intracellular tail. Yet there is only 60%identity in the extracellular loops (ECL), and 40% identity in theN-terminal domain, which has an additional sixteen amino acids.

[0229] All but one of the putative phosphorylation sites in CRF-RA₁ arefound in CRF-RB₁, the missing one being in the C-terminus. CRF-RB₁ alsohas an extra cysteine in a region of the N-terminus which may be withina cleaved putative signal peptide, as well as an extra cysteine at thejunction of extracellular loop-1 and transmembrane domain-3, so that theresidue may fall within this transmembrane domain.

Example 8 Pharmacologic Characteristics of CRF-RB₁ Transiently Expressedin COSM6 Cells

[0230] Using the methods described in Example 3, a radioreceptor assaywas conducted. Approximately 10 mg of pCRF-RB₁ plasmid DNA wastransfected into COSM6 cells using the DEAE dextran method. Two dayslater, the cells were detached and crude membrane fractions wereprepared and used to measure binding by competitive displacement of¹²⁵I-(Nle21,Tyr32)-ovine CRF. In order to calculate a K_(d), thedisplacement data were analyzed using the Ligand program of Munson andRodbard (1980), Anal. Biochem., 107:220-239.

[0231] The cloned CRF-RB₁ binds CRF with high affinity as determined bythe competitive displacement of bound radioligand. From six experiments,the dissociation constant was determined to be approximatelyK_(d)=1.3±0.2 nM (see FIG. 4). The binding is specific because thepeptides GRF and VIP do not displace the bound radioligand. In addition,the CRF-Rs have high affinity for sauvagine, urotensin, and potent CRFantagonists, such as [DPhe¹², Nle^(21,38)]-hCRF(9-41).

Example 9 Assay of CRF-RB₁ Mediated Stimulation of Intracellular cAMPLevels

[0232] Using methods described in Example 4, the ability of CRF-RB₁ tostimulate cAMP formation in CRF-RB₁-expressing COSM6 cells wasinvestigated. The plasmid PCRF-RB₁ was transfected into COSM6 cells. Oneday later, the cells were trypsinized and replated in 10% FBS, DMEM into24 or 48 well COSTAR tissue culture wells, and allowed to grow another24 hours. The medium was changed to 0.1% FBS, DMEM at least two hoursbefore treatments. The cells were preincubated for 30 minutes with 0.1mM 3-isobutyl-1-methylxanthine and then exposed to various peptides for30 minutes at 37° C. Intracellular cAMP was measured in duplicate fromtriplicate wells using an RIA kit (Biomedical Technologies, Stoughton,Mass.).

[0233] The results are presented in FIG. 5. The results indicate thatCRF stimulates the accumulation of intracellular cAMP when CRF-RB₁ istransiently transfected into COSM6 cells. The EC₅₀ occurs between 1-10nM, and the dose response is similar to that seen when the mouse analogof CRF-RA₁ is transfected. Urotensin and sauvagine, which are members ofthe CRF peptide family, are equipotent to CRF in stimulatingintracellular cAMP accumulation. The peptides GRF and VIP do notstimulate cAMP accumulation (see FIG. 5). The CRF signal transduced bythe cloned CRF-RB₁ receptor is inhibited in the presence of 1 mM CRFantagonist, (DPhe¹²,Nle^(21,38))hCRF(12-41).

Example 10 RNase Protection Assay to Determine Tissue Distribution ofCRF-RB₁

[0234] The coding region for mouse receptor corresponding to CRF-RA₁ wascloned by the well-known RT-PCR method using primers based on thepublished sequence, and RNA from mouse AtT-20 cells (ATCC No. CCl 89) astemplate. Plasmid DNAs encoding amino acids 26-106 of mouse CRF-RA₁ (SEQID NO:13) and amino acids 1 to 132 of mouse CRF-RB₁ (i.e., amino acids1-132 of SEQ ID NO:9) were linearized, and antisense riboprobes weresynthesized using SP6 RNA polymerase and [a-³²P]UTP. An internal loadingcontrol antisense riboprobe of glyceraldehyde 3-phosphate dehydrogenase(GAPDH) was synthesized using T7 RNA polymerase.

[0235] RNase protection assays were performed by hybridizing 30 mg oftotal RNA from mouse heart and brain tissues to 5×10⁵ cmp of labeledriboprobe at 65° C. for 18 hours. This was followed by RNase digestion(180 mg/ml RNase A and 350 U/ml RNase T1) at 23° C. for 60 minutes,after which samples were run on 5% polyacrylamide, 8 M urea gels.

[0236] RNase protection analysis was used to investigate the relativeexpression of CRF-RA₁ and CRF-RB₁ in mouse brain and heart. Whereas bothreceptors are detected in the brain and the heart, the heart appearsprimarily to express CRF-RB₁ (which has also been reported to expressCRF mRNA). There are multiple protected fragments seen in the heart.Furthermore, using a 5′ CRF-RB₁ probe, the major protected fragment inthe brain is smaller than in the heart, and smaller than that expectedto be generated from the cloned CRF-RB₁.

Example 11 In-Situ Hybridization Assay to Determine Tissue Distributionof CRF-RB₁

[0237] The tissue distribution of CRF-RB₁ mRNA was further characterizedby in situ hybridization histochemistry using ³⁵S-labeled antisense cRNAprobes. Six week old male C57BL/6 mice were perfused transcardially with4% paraformaldehyde in 0.1M borate buffer,and regularly spaced series of20-30 mm thick frozen sections through brain, heart, duodenum andtestis/epididymis were taken as described (Simmons et al., J.Histotechnology 12:169-181 (1989)). Radiolabeled antisense and sense(control) cRNA copies were synthesized from a 1.0 kb BamH1 digest ofCRF-RB₁ cDNA, encompassing 80 bp of the 5′ untranslated region and 926bp of the CRF-RB₁ coding sequence, subcloned into pBluescript KS vector(Stratagene, La Jolla, Calif.). ³²S-UTP was used as the radioactiveisotope for probe synthesis, and in situ hybridization was performed aspreviously described (Simmons et al., supra; and Imaki et al., BrainRes. 496:35-44 (1989)). Probes were labeled to specific activities of1-3 ×10⁹ dpm/mg, and hybridization was carried out under high stringencyconditions (50% formamide with final washes in 0.2×SSC at 70° C.).

[0238] Sense-strand cRNAs labeled to similar specific activities failedto show any suggestion of positive localizations when applied to tissuesections adjoining those in which antisense probes revealed robustsignals. Consistent with cloning and RNase protection assay data,CRF-RB₁ transcripts were detected in the heart, where labeling appearedmost. prominently over perivascular cells, as well as in the epicardium.In the male reproductive tract, CRF-RB₁ mRNA was localized principallyin stromal tissue of the epididymis, while labeling in testis was at ornear background levels. CRF-RB₁ mRNA signal over duodenum appeared as adense band of silver grains over the submucosal layer, and,additionally, over isolated non-epithelial cells at the base of thevilli.

[0239] In the brain, CRF-RB₁ mRNA displayed a rather restricteddistribution, which contrasts in extent and topography with that forCRF-RA₁ mRNA in rat. In the septal region, for example, CRF-RB₁ mRNA isexpressed in circumscribed aspects of the lateral septal nucleus, whilethe CRF-RA₁ transcript is seen over the medial septal complex. Othermajor sites of CRF-RB₁ mRNA expression in the forebrain includecircumscribed aspects of the olfactory bulb, preoptic region,hypothalamus, and amygdala.

[0240] In each of these areas, the pattern of CRF-RB₁ expression is seento be distinct from that of the CRF-RA₁ in rat brain. It appearsunlikely that major species differences in CRF-R distribution are atplay, since the mouse CRF-RB₁ probe employed here yielded similarpatterns of hybridization in mouse and rat brain.

[0241] While the invention has been described in detail with referenceto certain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

Summary of Sequences

[0242] Sequence ID No. 1 is the nucleic acid sequence (and the deducedamino acid sequence) of a cDNA encoding a human-derived CRF receptor ofthe present invention (i.e., hCRF-RA₁).

[0243] Sequence ID No. 2 is the deduced amino acid sequence of thehuman-derived CRF receptor set forth in Sequence ID No. 1.

[0244] Sequence ID No. 3 is the nucleic acid sequence (and the deducedamino acid sequence) of a splice variant cDNA insert encoding a 29 aminoacid insert portion of the human-derived CRF receptor of the presentinvention. The splice variant cDNA insert is located between nucleotides516-517 of Sequence ID No:1 (thereby producing CRF-RA₂).

[0245] Sequence ID No. 4 is the deduced amino acid sequence of thehuman-derived CRF receptor splice variant insert set forth in SequenceID No. 3. The splice variant amino acid insert is located between aminoacids 145-146 of SEQ ID NO:2.

[0246] Sequence ID No. 5 is the nucleic acid sequence (and the deducedamino acid sequence) of a cDNA encoding region of a rat-derived CRFreceptor of the present invention (i.e., rCRF-RA).

[0247] Sequence ID No. 6 is the deduced amino acid sequence of therat-derived CRF receptor set forth in Sequence ID No. 5.

[0248] Sequence ID No. 7 is the nucleic acid sequence (and the deducedamino acid sequence) of two exons (less the intervening intron sequence)of a partial genomic clone encoding a mouse-derived CRF receptor of thepresent invention (i.e., mCRF-RB₁).

[0249] Sequence ID No. 8 is the deduced amino acid sequence of thehuman-derived CRF receptor set forth in Sequence ID No. 7.

[0250] Sequence ID No. 9 is the nucleic acid sequence (and the deducedamino acid sequence) of a cDNA encoding a type-B mouse-derived CRFreceptor of the present invention (i.e., mCRF-RB₁).

[0251] Sequence ID No. 10 is the deduced amino acid sequence of themouse-derived CRF-RB receptor set forth in Sequence ID No. 9.

[0252] Sequence ID No. 11 is the “sense” probe described in Example 7.

[0253] Sequence ID No. 12 is the “antisense” probe described in Example7.

[0254] Sequence ID No. 13 is the amino acid sequence of themouse-derived CRF-RA₁ receptor.

[0255] Sequence ID No. 14 is the nucleotide sequence of a splice variantcDNA including an insert encoding a 29 amino acid portion of thehuman-derived CRF receptor of the present invention. The splice variantcDNA insert is located between nucleotides 516-517 of Sequence ID No:1(thereby producing CRF-RA₂).

[0256] Sequence ID No. 15 is the amino sequence of a splice variant CRF(CRF-RA₂) including a 29 amino acid portion of the human-derived CRFreceptor of the present invention. The splice variant amino acid insertis located between amino acids 145-146 of SEQ ID NO:2.

1 15 1 1495 DNA Homo sapiens CDS (82)...(1326) /product = “Humanpituitary CRF-receptor” /note= “This sequence is encoded by clone”CRF-R1“.” 1 cgagcccgca gccgcccgcc ggttcctctg ggatgtccgt aggacccgggcattcaggac 60 ggtagccgag cgagcccgag g atg gga ggg cac ccg cag ctc cgtctc gtc 111 Met Gly Gly His Pro Gln Leu Arg Leu Val 1 5 10 aag gcc cttctc ctt ctg ggg ctg aac ccc gtc tct gcc tcc ctc cag 159 Lys Ala Leu LeuLeu Leu Gly Leu Asn Pro Val Ser Ala Ser Leu Gln 15 20 25 gac cag cac tgcgag agc ctg tcc ctg gcc agc aac atc tca gga ctg 207 Asp Gln His Cys GluSer Leu Ser Leu Ala Ser Asn Ile Ser Gly Leu 30 35 40 cag tgc aac gca tccgtg gac ctc att ggc acc tgc tgg ccc cgc agc 255 Gln Cys Asn Ala Ser ValAsp Leu Ile Gly Thr Cys Trp Pro Arg Ser 45 50 55 cct gcg ggg cag cta gtggtt cgg ccc tgc cct gcc ttt ttc tat ggt 303 Pro Ala Gly Gln Leu Val ValArg Pro Cys Pro Ala Phe Phe Tyr Gly 60 65 70 gtc cgc tac aat acc aca aacaat ggc tac cgg gag tgc ctg gcc aat 351 Val Arg Tyr Asn Thr Thr Asn AsnGly Tyr Arg Glu Cys Leu Ala Asn 75 80 85 90 ggc agc tgg gcc gcc cgc gtgaat tac tcc gag tgc cag gag atc ctc 399 Gly Ser Trp Ala Ala Arg Val AsnTyr Ser Glu Cys Gln Glu Ile Leu 95 100 105 aat gag gag aaa aaa agc aaggtg cac tac cat gtc gca gtc atc atc 447 Asn Glu Glu Lys Lys Ser Lys ValHis Tyr His Val Ala Val Ile Ile 110 115 120 aac tac ctg ggc cac tgt atctcc ctg gtg gcc ctc ctg gtg gcc ttt 495 Asn Tyr Leu Gly His Cys Ile SerLeu Val Ala Leu Leu Val Ala Phe 125 130 135 gtc ctc ttt ctg cgg ctc aggagc atc cgg tgc ctg cga aac atc atc 543 Val Leu Phe Leu Arg Leu Arg SerIle Arg Cys Leu Arg Asn Ile Ile 140 145 150 cac tgg aac ctc atc tcc gccttc atc ctg cgc aac gcc acc tgg ttc 591 His Trp Asn Leu Ile Ser Ala PheIle Leu Arg Asn Ala Thr Trp Phe 155 160 165 170 gtg gtc cag cta acc atgagc ccc gag gtc cac cag agc aac gtg ggc 639 Val Val Gln Leu Thr Met SerPro Glu Val His Gln Ser Asn Val Gly 175 180 185 tgg tgc agg ttg gtg acagcc gcc tac aac tac ttc cat gtg acc aac 687 Trp Cys Arg Leu Val Thr AlaAla Tyr Asn Tyr Phe His Val Thr Asn 190 195 200 ttc ttc tgg atg ttc ggcgag ggc tgc tac ctg cac aca gcc atc gtg 735 Phe Phe Trp Met Phe Gly GluGly Cys Tyr Leu His Thr Ala Ile Val 205 210 215 ctc acc tac tcc act gaccgg ctg cgc aaa tgg atg ttc atc tgc att 783 Leu Thr Tyr Ser Thr Asp ArgLeu Arg Lys Trp Met Phe Ile Cys Ile 220 225 230 ggc tgg ggt gtg ccc ttcccc atc att gtg gcc tgg gcc att ggg aag 831 Gly Trp Gly Val Pro Phe ProIle Ile Val Ala Trp Ala Ile Gly Lys 235 240 245 250 ctg tac tac gac aatgag aag tgc tgg ttt ggc aaa agg cct ggg gtg 879 Leu Tyr Tyr Asp Asn GluLys Cys Trp Phe Gly Lys Arg Pro Gly Val 255 260 265 tac acc gac tac atctac cag ggc ccc atg atc ctg gtc ctg ctg atc 927 Tyr Thr Asp Tyr Ile TyrGln Gly Pro Met Ile Leu Val Leu Leu Ile 270 275 280 aat ttc atc ttc cttttc aac atc gtc cgc atc ctc atg acc aag ctc 975 Asn Phe Ile Phe Leu PheAsn Ile Val Arg Ile Leu Met Thr Lys Leu 285 290 295 cgg gca tcc acc acgtct gag acc att cag tac agg aag gct gtg aaa 1023 Arg Ala Ser Thr Thr SerGlu Thr Ile Gln Tyr Arg Lys Ala Val Lys 300 305 310 gcc act ctg gtg ctgctg ccc ctc ctg ggc atc acc tac atg ctg ttc 1071 Ala Thr Leu Val Leu LeuPro Leu Leu Gly Ile Thr Tyr Met Leu Phe 315 320 325 330 ttc gtc aat cccggg gag gat gag gtc tcc cgg gtc gtc ttc atc tac 1119 Phe Val Asn Pro GlyGlu Asp Glu Val Ser Arg Val Val Phe Ile Tyr 335 340 345 ttc aac tcc ttcctg gaa tcc ttc cag ggc ttc ttt gtg tct gtg ttc 1167 Phe Asn Ser Phe LeuGlu Ser Phe Gln Gly Phe Phe Val Ser Val Phe 350 355 360 tac tgt ttc ctcaat agt gag gtc cgt tct gcc atc cgg aag agg tgg 1215 Tyr Cys Phe Leu AsnSer Glu Val Arg Ser Ala Ile Arg Lys Arg Trp 365 370 375 cac cgg tgg caggac aag cac tcg atc cgt gcc cga gtg gcc cgt gcc 1263 His Arg Trp Gln AspLys His Ser Ile Arg Ala Arg Val Ala Arg Ala 380 385 390 atg tcc atc cccacc tcc cca acc cgt gtc agc ttt cac agc atc aag 1311 Met Ser Ile Pro ThrSer Pro Thr Arg Val Ser Phe His Ser Ile Lys 395 400 405 410 cag tcc acagca gtc tgagctggca ggtcatggag cagcccccaa agagctgtgg 1366 Gln Ser Thr AlaVal 415 ctggggggat gacggccagg ctccctgacc accctgcctg tggaggtgacctgttaggtc 1426 tcatgcccac tcccccagga gcagctggca ctgacagcct gggggggccgctctccccct 1486 gcagccgtg 1495 2 415 PRT Homo sapiens 2 Met Gly Gly HisPro Gln Leu Arg Leu Val Lys Ala Leu Leu Leu Leu 1 5 10 15 Gly Leu AsnPro Val Ser Ala Ser Leu Gln Asp Gln His Cys Glu Ser 20 25 30 Leu Ser LeuAla Ser Asn Ile Ser Gly Leu Gln Cys Asn Ala Ser Val 35 40 45 Asp Leu IleGly Thr Cys Trp Pro Arg Ser Pro Ala Gly Gln Leu Val 50 55 60 Val Arg ProCys Pro Ala Phe Phe Tyr Gly Val Arg Tyr Asn Thr Thr 65 70 75 80 Asn AsnGly Tyr Arg Glu Cys Leu Ala Asn Gly Ser Trp Ala Ala Arg 85 90 95 Val AsnTyr Ser Glu Cys Gln Glu Ile Leu Asn Glu Glu Lys Lys Ser 100 105 110 LysVal His Tyr His Val Ala Val Ile Ile Asn Tyr Leu Gly His Cys 115 120 125Ile Ser Leu Val Ala Leu Leu Val Ala Phe Val Leu Phe Leu Arg Leu 130 135140 Arg Ser Ile Arg Cys Leu Arg Asn Ile Ile His Trp Asn Leu Ile Ser 145150 155 160 Ala Phe Ile Leu Arg Asn Ala Thr Trp Phe Val Val Gln Leu ThrMet 165 170 175 Ser Pro Glu Val His Gln Ser Asn Val Gly Trp Cys Arg LeuVal Thr 180 185 190 Ala Ala Tyr Asn Tyr Phe His Val Thr Asn Phe Phe TrpMet Phe Gly 195 200 205 Glu Gly Cys Tyr Leu His Thr Ala Ile Val Leu ThrTyr Ser Thr Asp 210 215 220 Arg Leu Arg Lys Trp Met Phe Ile Cys Ile GlyTrp Gly Val Pro Phe 225 230 235 240 Pro Ile Ile Val Ala Trp Ala Ile GlyLys Leu Tyr Tyr Asp Asn Glu 245 250 255 Lys Cys Trp Phe Gly Lys Arg ProGly Val Tyr Thr Asp Tyr Ile Tyr 260 265 270 Gln Gly Pro Met Ile Leu ValLeu Leu Ile Asn Phe Ile Phe Leu Phe 275 280 285 Asn Ile Val Arg Ile LeuMet Thr Lys Leu Arg Ala Ser Thr Thr Ser 290 295 300 Glu Thr Ile Gln TyrArg Lys Ala Val Lys Ala Thr Leu Val Leu Leu 305 310 315 320 Pro Leu LeuGly Ile Thr Tyr Met Leu Phe Phe Val Asn Pro Gly Glu 325 330 335 Asp GluVal Ser Arg Val Val Phe Ile Tyr Phe Asn Ser Phe Leu Glu 340 345 350 SerPhe Gln Gly Phe Phe Val Ser Val Phe Tyr Cys Phe Leu Asn Ser 355 360 365Glu Val Arg Ser Ala Ile Arg Lys Arg Trp His Arg Trp Gln Asp Lys 370 375380 His Ser Ile Arg Ala Arg Val Ala Arg Ala Met Ser Ile Pro Thr Ser 385390 395 400 Pro Thr Arg Val Ser Phe His Ser Ile Lys Gln Ser Thr Ala Val405 410 415 3 87 DNA Homo sapiens CDS (1)...(87) CRF-R splice variantinsert fragment 3 cca ggc tgc acc cat tgg ggt gac cag gca gat gga gccctg gag gtg 48 Pro Gly Cys Thr His Trp Gly Asp Gln Ala Asp Gly Ala LeuGlu Val 1 5 10 15 ggg gct cca tgg agt ggt gcc cca ttt cag gtt cga agg 87Gly Ala Pro Trp Ser Gly Ala Pro Phe Gln Val Arg Arg 20 25 4 29 PRT Homosapiens 4 Pro Gly Cys Thr His Trp Gly Asp Gln Ala Asp Gly Ala Leu GluVal 1 5 10 15 Gly Ala Pro Trp Ser Gly Ala Pro Phe Gln Val Arg Arg 20 255 1411 DNA Rattus CDS (80)...(1324) 5 agaccgcagc cgcccgccct ccgctctgggatgtcggagc gatccaggca tccaggacgc 60 tgacggagcg agcccgagg atg gga cgg cgcccg cag ctc cgg ctc gtg aag 112 Met Gly Arg Arg Pro Gln Leu Arg Leu ValLys 1 5 10 gcc ctt ctc ctt ctg ggg ctg aac cct gtg tcc acc tcc ctt caggat 160 Ala Leu Leu Leu Leu Gly Leu Asn Pro Val Ser Thr Ser Leu Gln Asp15 20 25 cag cgc tgt gag aac ctg tcc ctg acc agc aat gtt tct ggc ctg cag208 Gln Arg Cys Glu Asn Leu Ser Leu Thr Ser Asn Val Ser Gly Leu Gln 3035 40 tgc aat gca tcc gtg gac ctc att ggc acc tgc tgg ccc cgg agc cct256 Cys Asn Ala Ser Val Asp Leu Ile Gly Thr Cys Trp Pro Arg Ser Pro 4550 55 gcg ggc cag ttg gtg gtc cga ccc tgc cct gcc ttt ttc tac ggt gtc304 Ala Gly Gln Leu Val Val Arg Pro Cys Pro Ala Phe Phe Tyr Gly Val 6065 70 75 cgc tac aac acg aca aac aat ggc tac cgg gag tgc ctg gcc aac ggc352 Arg Tyr Asn Thr Thr Asn Asn Gly Tyr Arg Glu Cys Leu Ala Asn Gly 8085 90 agc tgg gca gcc cgt gtg aat tat tct gag tgc cag gag att ctc aac400 Ser Trp Ala Ala Arg Val Asn Tyr Ser Glu Cys Gln Glu Ile Leu Asn 95100 105 gaa gag aag aag agc aaa gta cac tac cat gtt gca gtc atc atc aac448 Glu Glu Lys Lys Ser Lys Val His Tyr His Val Ala Val Ile Ile Asn 110115 120 tac ctg ggt cac tgc atc tcc ctg gta gcc ctc ctg gtg gcc ttt gtc496 Tyr Leu Gly His Cys Ile Ser Leu Val Ala Leu Leu Val Ala Phe Val 125130 135 ctc ttc ttg cgg ctc agg agc atc cgg tgc ctg aga aac atc atc cac544 Leu Phe Leu Arg Leu Arg Ser Ile Arg Cys Leu Arg Asn Ile Ile His 140145 150 155 tgg aac ctc atc tcg gct ttc atc cta cgc aac gcc acg tgg tttgtg 592 Trp Asn Leu Ile Ser Ala Phe Ile Leu Arg Asn Ala Thr Trp Phe Val160 165 170 gtc cag ctc acc gtg agc ccc gag gtg cac cag agc aat gtg gcctgg 640 Val Gln Leu Thr Val Ser Pro Glu Val His Gln Ser Asn Val Ala Trp175 180 185 tgt agg ttg gtg aca gcc gcc tac aat tac ttc cat gta acc aacttc 688 Cys Arg Leu Val Thr Ala Ala Tyr Asn Tyr Phe His Val Thr Asn Phe190 195 200 ttc tgg atg ttc ggt gag ggc tgc tac ctg cac aca gcc att gtgctc 736 Phe Trp Met Phe Gly Glu Gly Cys Tyr Leu His Thr Ala Ile Val Leu205 210 215 acg tac tcc acc gac cgt ctg cgc aag tgg atg ttc gtc tgc attggc 784 Thr Tyr Ser Thr Asp Arg Leu Arg Lys Trp Met Phe Val Cys Ile Gly220 225 230 235 tgg ggt gta cct ttc ccc atc att gtg gct tgg gcc att gggaag ctg 832 Trp Gly Val Pro Phe Pro Ile Ile Val Ala Trp Ala Ile Gly LysLeu 240 245 250 cac tac gac aat gaa aag tgc tgg ttt ggc aaa cgt cct ggggta tac 880 His Tyr Asp Asn Glu Lys Cys Trp Phe Gly Lys Arg Pro Gly ValTyr 255 260 265 act gac tac atc tac cag ggc ccc atg atc ctg gtc ctg ctgatc aac 928 Thr Asp Tyr Ile Tyr Gln Gly Pro Met Ile Leu Val Leu Leu IleAsn 270 275 280 ttt atc ttt ctc ttc aac att gtc cgc atc ctc atg acc aaactc cgg 976 Phe Ile Phe Leu Phe Asn Ile Val Arg Ile Leu Met Thr Lys LeuArg 285 290 295 gca tcc act aca tct gag acc att cag tac agg aag gct gtgaag gcc 1024 Ala Ser Thr Thr Ser Glu Thr Ile Gln Tyr Arg Lys Ala Val LysAla 300 305 310 315 act ctg gtg ctc ctg ccc ctt ctg ggc atc acc tac atgttg ttc ttc 1072 Thr Leu Val Leu Leu Pro Leu Leu Gly Ile Thr Tyr Met LeuPhe Phe 320 325 330 gtc aac cct gga gag gac gag gtc tcc agg gtc gtc ttcatc tac ttc 1120 Val Asn Pro Gly Glu Asp Glu Val Ser Arg Val Val Phe IleTyr Phe 335 340 345 aac tct ttt ctg gag tcc ttt cag ggc ttc ttt gtg tctgtg ttc tac 1168 Asn Ser Phe Leu Glu Ser Phe Gln Gly Phe Phe Val Ser ValPhe Tyr 350 355 360 tgt ttt ctg aac agt gag gtc cgc tcc gct atc cgg aagagg tgg cgt 1216 Cys Phe Leu Asn Ser Glu Val Arg Ser Ala Ile Arg Lys ArgTrp Arg 365 370 375 cgg tgg cag gac aag cac tcc atc aga gcc cga gtg gcccga gct atg 1264 Arg Trp Gln Asp Lys His Ser Ile Arg Ala Arg Val Ala ArgAla Met 380 385 390 395 tcc atc ccc acc tcc ccg acc aga gtc agc ttt cacagc atc aag cag 1312 Ser Ile Pro Thr Ser Pro Thr Arg Val Ser Phe His SerIle Lys Gln 400 405 410 tcc aca gca gtg tgagctccag gccacagagc agcccccaagacctgaggcc 1364 Ser Thr Ala Val 415 ggggagatga tgcaagctca ctgacgagccagtctgcaga cgcaagc 1411 6 415 PRT Rattus 6 Met Gly Arg Arg Pro Gln LeuArg Leu Val Lys Ala Leu Leu Leu Leu 1 5 10 15 Gly Leu Asn Pro Val SerThr Ser Leu Gln Asp Gln Arg Cys Glu Asn 20 25 30 Leu Ser Leu Thr Ser AsnVal Ser Gly Leu Gln Cys Asn Ala Ser Val 35 40 45 Asp Leu Ile Gly Thr CysTrp Pro Arg Ser Pro Ala Gly Gln Leu Val 50 55 60 Val Arg Pro Cys Pro AlaPhe Phe Tyr Gly Val Arg Tyr Asn Thr Thr 65 70 75 80 Asn Asn Gly Tyr ArgGlu Cys Leu Ala Asn Gly Ser Trp Ala Ala Arg 85 90 95 Val Asn Tyr Ser GluCys Gln Glu Ile Leu Asn Glu Glu Lys Lys Ser 100 105 110 Lys Val His TyrHis Val Ala Val Ile Ile Asn Tyr Leu Gly His Cys 115 120 125 Ile Ser LeuVal Ala Leu Leu Val Ala Phe Val Leu Phe Leu Arg Leu 130 135 140 Arg SerIle Arg Cys Leu Arg Asn Ile Ile His Trp Asn Leu Ile Ser 145 150 155 160Ala Phe Ile Leu Arg Asn Ala Thr Trp Phe Val Val Gln Leu Thr Val 165 170175 Ser Pro Glu Val His Gln Ser Asn Val Ala Trp Cys Arg Leu Val Thr 180185 190 Ala Ala Tyr Asn Tyr Phe His Val Thr Asn Phe Phe Trp Met Phe Gly195 200 205 Glu Gly Cys Tyr Leu His Thr Ala Ile Val Leu Thr Tyr Ser ThrAsp 210 215 220 Arg Leu Arg Lys Trp Met Phe Val Cys Ile Gly Trp Gly ValPro Phe 225 230 235 240 Pro Ile Ile Val Ala Trp Ala Ile Gly Lys Leu HisTyr Asp Asn Glu 245 250 255 Lys Cys Trp Phe Gly Lys Arg Pro Gly Val TyrThr Asp Tyr Ile Tyr 260 265 270 Gln Gly Pro Met Ile Leu Val Leu Leu IleAsn Phe Ile Phe Leu Phe 275 280 285 Asn Ile Val Arg Ile Leu Met Thr LysLeu Arg Ala Ser Thr Thr Ser 290 295 300 Glu Thr Ile Gln Tyr Arg Lys AlaVal Lys Ala Thr Leu Val Leu Leu 305 310 315 320 Pro Leu Leu Gly Ile ThrTyr Met Leu Phe Phe Val Asn Pro Gly Glu 325 330 335 Asp Glu Val Ser ArgVal Val Phe Ile Tyr Phe Asn Ser Phe Leu Glu 340 345 350 Ser Phe Gln GlyPhe Phe Val Ser Val Phe Tyr Cys Phe Leu Asn Ser 355 360 365 Glu Val ArgSer Ala Ile Arg Lys Arg Trp Arg Arg Trp Gln Asp Lys 370 375 380 His SerIle Arg Ala Arg Val Ala Arg Ala Met Ser Ile Pro Thr Ser 385 390 395 400Pro Thr Arg Val Ser Phe His Ser Ile Lys Gln Ser Thr Ala Val 405 410 4157 210 DNA Mus musculus CDS (1)...(210) 7 tgg tgc cgc tgc atc acc acc atcttc aac tat ttt gtg gtc acc aac 48 Trp Cys Arg Cys Ile Thr Thr Ile PheAsn Tyr Phe Val Val Thr Asn 1 5 10 15 ttc ttc tgg atg ttt gtg gag gggtgc tac ctg cac acg gcc att gtc 96 Phe Phe Trp Met Phe Val Glu Gly CysTyr Leu His Thr Ala Ile Val 20 25 30 atg acg tac tcc aca gag cac ctg cgcaag tgg ctt ttc ctc ttc att 144 Met Thr Tyr Ser Thr Glu His Leu Arg LysTrp Leu Phe Leu Phe Ile 35 40 45 gga tgg tgc att ccc tgc cct atc atc atcgcc tgg gca gtt ggc aaa 192 Gly Trp Cys Ile Pro Cys Pro Ile Ile Ile AlaTrp Ala Val Gly Lys 50 55 60 ctc tac tat gag aat gag 210 Leu Tyr Tyr GluAsn Glu 65 70 8 70 PRT Mus musculus 8 Trp Cys Arg Cys Ile Thr Thr IlePhe Asn Tyr Phe Val Val Thr Asn 1 5 10 15 Phe Phe Trp Met Phe Val GluGly Cys Tyr Leu His Thr Ala Ile Val 20 25 30 Met Thr Tyr Ser Thr Glu HisLeu Arg Lys Trp Leu Phe Leu Phe Ile 35 40 45 Gly Trp Cys Ile Pro Cys ProIle Ile Ile Ala Trp Ala Val Gly Lys 50 55 60 Leu Tyr Tyr Glu Asn Glu 6570 9 1374 DNA Mus musculus CDS (79)...(1371) 9 gccggacaga cctcctttggaagcagccac ttctggtccc catccctgga gcgatcgagc 60 ggcaggatct gctgtccc atgggg acc cca ggc tct ctt ccc agt gca cag 111 Met Gly Thr Pro Gly Ser LeuPro Ser Ala Gln 1 5 10 ctt ctc ctc tgc ctg ttt tcc ctg ctt cca gtg ctccag gtg gcc caa 159 Leu Leu Leu Cys Leu Phe Ser Leu Leu Pro Val Leu GlnVal Ala Gln 15 20 25 cca ggc cag gca ccc cag gac cag ccc ctg tgg aca cttttg gag cag 207 Pro Gly Gln Ala Pro Gln Asp Gln Pro Leu Trp Thr Leu LeuGlu Gln 30 35 40 tac tgc cac agg acc aca att ggg aat ttt tca ggt ccc tacacc tac 255 Tyr Cys His Arg Thr Thr Ile Gly Asn Phe Ser Gly Pro Tyr ThrTyr 45 50 55 tgc aac acg acc ttg gac cag atc ggg acc tgc tgg cca cag agcgca 303 Cys Asn Thr Thr Leu Asp Gln Ile Gly Thr Cys Trp Pro Gln Ser Ala60 65 70 75 ccc gga gcc cta gta gag aga ccg tgc ccc gag tac ttc aat ggcatc 351 Pro Gly Ala Leu Val Glu Arg Pro Cys Pro Glu Tyr Phe Asn Gly Ile80 85 90 aag tac aac acg acc cgg aat gcc tac aga gag tgc ctg gag aac ggg399 Lys Tyr Asn Thr Thr Arg Asn Ala Tyr Arg Glu Cys Leu Glu Asn Gly 95100 105 acc tgg gcc tca agg gtc aac tac tca cac tgc gaa ccc att ttg gat447 Thr Trp Ala Ser Arg Val Asn Tyr Ser His Cys Glu Pro Ile Leu Asp 110115 120 gac aag cag aga aag tat gac ctg cat tac cga atc gcc ctc att gtc495 Asp Lys Gln Arg Lys Tyr Asp Leu His Tyr Arg Ile Ala Leu Ile Val 125130 135 aac tac ctg ggt cac tgt gtt tcc gtg gtg gcc ctg gtg gcc gct ttc543 Asn Tyr Leu Gly His Cys Val Ser Val Val Ala Leu Val Ala Ala Phe 140145 150 155 ctg ctt ttc cta gtg ctg cgg agt atc cgc tgc ctg agg aat gtgatc 591 Leu Leu Phe Leu Val Leu Arg Ser Ile Arg Cys Leu Arg Asn Val Ile160 165 170 cac tgg aac ctc atc acc acc ttc att ctg aga aac atc gcg tggttc 639 His Trp Asn Leu Ile Thr Thr Phe Ile Leu Arg Asn Ile Ala Trp Phe175 180 185 ctg ctg caa ctc atc gac cac gaa gtg cac gag ggc aat gag gtctgg 687 Leu Leu Gln Leu Ile Asp His Glu Val His Glu Gly Asn Glu Val Trp190 195 200 tgc cgc tgc atc acc acc atc ttc aac tat ttt gtg gtc acc aacttc 735 Cys Arg Cys Ile Thr Thr Ile Phe Asn Tyr Phe Val Val Thr Asn Phe205 210 215 ttc tgg atg ttt gtg gag ggc tgc tac ctg cac acg gcc att gtcatg 783 Phe Trp Met Phe Val Glu Gly Cys Tyr Leu His Thr Ala Ile Val Met220 225 230 235 acg tac tcc aca gag cac ctg cgc aag tgg ctt ttc ctc ttcatt gga 831 Thr Tyr Ser Thr Glu His Leu Arg Lys Trp Leu Phe Leu Phe IleGly 240 245 250 tgg tgc att ccc tgc cct atc atc atc gcc tgg gca gtt ggcaaa ctc 879 Trp Cys Ile Pro Cys Pro Ile Ile Ile Ala Trp Ala Val Gly LysLeu 255 260 265 tac tat gag aat gag cag tgc tgg ttt ggc aag gaa gct ggtgat ttg 927 Tyr Tyr Glu Asn Glu Gln Cys Trp Phe Gly Lys Glu Ala Gly AspLeu 270 275 280 gtg gac tac atc tac cag ggc ccc gtc atg ctt gtg ctg ttgatc aat 975 Val Asp Tyr Ile Tyr Gln Gly Pro Val Met Leu Val Leu Leu IleAsn 285 290 295 ttt gta ttt ctg ttt aac atc gtc agg atc ctg atg acg aagtta cga 1023 Phe Val Phe Leu Phe Asn Ile Val Arg Ile Leu Met Thr Lys LeuArg 300 305 310 315 gca tcc acc acg tcc gag aca atc caa tac agg aag gcagtg aag gcc 1071 Ala Ser Thr Thr Ser Glu Thr Ile Gln Tyr Arg Lys Ala ValLys Ala 320 325 330 acg ctg gtc ctc ctc ccc ctg ttg ggc atc acc tac atgctc ttc ttt 1119 Thr Leu Val Leu Leu Pro Leu Leu Gly Ile Thr Tyr Met LeuPhe Phe 335 340 345 gtc aat cct ggc gag gac gac ctg tcc cag att gtg ttcatc tac ttc 1167 Val Asn Pro Gly Glu Asp Asp Leu Ser Gln Ile Val Phe IleTyr Phe 350 355 360 aac tct ttc ctg cag tcc ttc cag ggt ttc ttt gtg tccgtt ttc tac 1215 Asn Ser Phe Leu Gln Ser Phe Gln Gly Phe Phe Val Ser ValPhe Tyr 365 370 375 tgc ttc ttc aat gga gag gtg cgc gcg gcc ctg aga aagcgg tgg cac 1263 Cys Phe Phe Asn Gly Glu Val Arg Ala Ala Leu Arg Lys ArgTrp His 380 385 390 395 cgc tgg cag gac cac cac gcc ctc cgg gtg cct gtggcc cgg gcc atg 1311 Arg Trp Gln Asp His His Ala Leu Arg Val Pro Val AlaArg Ala Met 400 405 410 tcc atc cct acg tcg ccc acc agg atc agc ttc cacagc atc aag cag 1359 Ser Ile Pro Thr Ser Pro Thr Arg Ile Ser Phe His SerIle Lys Gln 415 420 425 aca gct gct gtg tga 1374 Thr Ala Ala Val 430 10431 PRT Mus musculus 10 Met Gly Thr Pro Gly Ser Leu Pro Ser Ala Gln LeuLeu Leu Cys Leu 1 5 10 15 Phe Ser Leu Leu Pro Val Leu Gln Val Ala GlnPro Gly Gln Ala Pro 20 25 30 Gln Asp Gln Pro Leu Trp Thr Leu Leu Glu GlnTyr Cys His Arg Thr 35 40 45 Thr Ile Gly Asn Phe Ser Gly Pro Tyr Thr TyrCys Asn Thr Thr Leu 50 55 60 Asp Gln Ile Gly Thr Cys Trp Pro Gln Ser AlaPro Gly Ala Leu Val 65 70 75 80 Glu Arg Pro Cys Pro Glu Tyr Phe Asn GlyIle Lys Tyr Asn Thr Thr 85 90 95 Arg Asn Ala Tyr Arg Glu Cys Leu Glu AsnGly Thr Trp Ala Ser Arg 100 105 110 Val Asn Tyr Ser His Cys Glu Pro IleLeu Asp Asp Lys Gln Arg Lys 115 120 125 Tyr Asp Leu His Tyr Arg Ile AlaLeu Ile Val Asn Tyr Leu Gly His 130 135 140 Cys Val Ser Val Val Ala LeuVal Ala Ala Phe Leu Leu Phe Leu Val 145 150 155 160 Leu Arg Ser Ile ArgCys Leu Arg Asn Val Ile His Trp Asn Leu Ile 165 170 175 Thr Thr Phe IleLeu Arg Asn Ile Ala Trp Phe Leu Leu Gln Leu Ile 180 185 190 Asp His GluVal His Glu Gly Asn Glu Val Trp Cys Arg Cys Ile Thr 195 200 205 Thr IlePhe Asn Tyr Phe Val Val Thr Asn Phe Phe Trp Met Phe Val 210 215 220 GluGly Cys Tyr Leu His Thr Ala Ile Val Met Thr Tyr Ser Thr Glu 225 230 235240 His Leu Arg Lys Trp Leu Phe Leu Phe Ile Gly Trp Cys Ile Pro Cys 245250 255 Pro Ile Ile Ile Ala Trp Ala Val Gly Lys Leu Tyr Tyr Glu Asn Glu260 265 270 Gln Cys Trp Phe Gly Lys Glu Ala Gly Asp Leu Val Asp Tyr IleTyr 275 280 285 Gln Gly Pro Val Met Leu Val Leu Leu Ile Asn Phe Val PheLeu Phe 290 295 300 Asn Ile Val Arg Ile Leu Met Thr Lys Leu Arg Ala SerThr Thr Ser 305 310 315 320 Glu Thr Ile Gln Tyr Arg Lys Ala Val Lys AlaThr Leu Val Leu Leu 325 330 335 Pro Leu Leu Gly Ile Thr Tyr Met Leu PhePhe Val Asn Pro Gly Glu 340 345 350 Asp Asp Leu Ser Gln Ile Val Phe IleTyr Phe Asn Ser Phe Leu Gln 355 360 365 Ser Phe Gln Gly Phe Phe Val SerVal Phe Tyr Cys Phe Phe Asn Gly 370 375 380 Glu Val Arg Ala Ala Leu ArgLys Arg Trp His Arg Trp Gln Asp His 385 390 395 400 His Ala Leu Arg ValPro Val Ala Arg Ala Met Ser Ile Pro Thr Ser 405 410 415 Pro Thr Arg IleSer Phe His Ser Ile Lys Gln Thr Ala Ala Val 420 425 430 11 23 DNAArtificial Sequence “sense” probe for CRF-RB1 11 ctgcatcacc accatcttcaact 23 12 20 DNA Artificial Sequence “antisense” probe for CRF-RB1 12agccacttgc gcaggtgctc 20 13 415 PRT Mus musucus 13 Met Gly Gln Arg ProGln Leu Arg Leu Val Lys Ala Leu Leu Leu Leu 1 5 10 15 Gly Leu Asn ProVal Ser Thr Ser Leu Gln Asp Gln Gln Cys Glu Ser 20 25 30 Leu Ser Leu AlaSer Asn Val Ser Gly Leu Gln Cys Asn Ala Ser Val 35 40 45 Asp Leu Ile GlyThr Cys Trp Pro Arg Ser Pro Ala Gly Gln Leu Val 50 55 60 Val Arg Pro CysPro Ala Phe Phe Tyr Gly Val Arg Tyr Asn Thr Thr 65 70 75 80 Asn Asn GlyTyr Arg Glu Cys Leu Ala Asn Gly Ser Trp Ala Ala Arg 85 90 95 Val Asn TyrSer Glu Cys Gln Glu Ile Leu Asn Glu Glu Lys Lys Ser 100 105 110 Lys ValHis Tyr His Ile Ala Val Ile Ile Asn Tyr Leu Gly His Cys 115 120 125 IleSer Leu Val Ala Leu Leu Val Ala Phe Val Leu Phe Leu Arg Leu 130 135 140Arg Ser Ile Arg Cys Leu Arg Asn Ile Ile His Trp Asn Leu Ile Ser 145 150155 160 Ala Phe Ile Leu Arg Asn Ala Thr Trp Phe Val Val Gln Leu Thr Val165 170 175 Ser Pro Glu Val His Gln Ser Asn Val Ala Trp Cys Arg Leu ValThr 180 185 190 Ala Ala Tyr Asn Tyr Phe His Val Thr Asn Phe Phe Trp MetPhe Gly 195 200 205 Glu Gly Cys Tyr Leu His Thr Ala Ile Val Leu Thr TyrSer Thr Asp 210 215 220 Arg Leu Arg Lys Trp Met Phe Val Cys Ile Gly TrpGly Val Pro Phe 225 230 235 240 Pro Ile Ile Val Ala Trp Ala Ile Gly LysLeu Tyr Tyr Asp Asn Glu 245 250 255 Lys Cys Trp Phe Gly Lys Arg Pro GlyVal Tyr Thr Asp Tyr Ile Tyr 260 265 270 Gln Gly Pro Met Ile Leu Val LeuLeu Ile Asn Phe Ile Phe Leu Phe 275 280 285 Asn Ile Val Arg Ile Leu MetThr Lys Leu Arg Ala Ser Thr Thr Ser 290 295 300 Glu Thr Ile Gln Tyr ArgLys Ala Val Lys Ala Thr Leu Val Leu Leu 305 310 315 320 Pro Leu Leu GlyIle Thr Tyr Met Leu Phe Phe Val Asn Pro Gly Glu 325 330 335 Asp Glu ValSer Arg Val Val Phe Ile Tyr Phe Asn Ser Phe Leu Glu 340 345 350 Ser PheGln Gly Phe Phe Val Ser Val Phe Tyr Cys Phe Leu Asn Ser 355 360 365 GluVal Arg Ser Ala Ile Arg Lys Arg Trp Arg Arg Trp Gln Asp Lys 370 375 380His Ser Ile Arg Ala Arg Val Ala Arg Ala Met Ser Ile Pro Thr Ser 385 390395 400 Pro Thr Arg Val Ser Phe His Ser Ile Lys Gln Ser Thr Ala Val 405410 415 14 1582 DNA Homo sapiens CDS (82)...(1413) CRF-R splice-variantinsert fragment inserted between nucleotides 516-517 of SEQ ID NO1./note= “This sequence is contained in clone ”CRF-R2“.” 14 cgagcccgcagccgcccgcc ggttcctctg ggatgtccgt aggacccggg cattcaggac 60 ggtagccgagcgagcccgag g atg gga ggg cac ccg cag ctc cgt ctc gtc 111 Met Gly Gly HisPro Gln Leu Arg Leu Val 1 5 10 aag gcc ctt ctc ctt ctg ggg ctg aac cccgtc tct gcc tcc ctc cag 159 Lys Ala Leu Leu Leu Leu Gly Leu Asn Pro ValSer Ala Ser Leu Gln 15 20 25 gac cag cac tgc gag agc ctg tcc ctg gcc agcaac atc tca gga ctg 207 Asp Gln His Cys Glu Ser Leu Ser Leu Ala Ser AsnIle Ser Gly Leu 30 35 40 cag tgc aac gca tcc gtg gac ctc att ggc acc tgctgg ccc cgc agc 255 Gln Cys Asn Ala Ser Val Asp Leu Ile Gly Thr Cys TrpPro Arg Ser 45 50 55 cct gcg ggg cag cta gtg gtt cgg ccc tgc cct gcc tttttc tat ggt 303 Pro Ala Gly Gln Leu Val Val Arg Pro Cys Pro Ala Phe PheTyr Gly 60 65 70 gtc cgc tac aat acc aca aac aat ggc tac cgg gag tgc ctggcc aat 351 Val Arg Tyr Asn Thr Thr Asn Asn Gly Tyr Arg Glu Cys Leu AlaAsn 75 80 85 90 ggc agc tgg gcc gcc cgc gtg aat tac tcc gag tgc cag gagatc ctc 399 Gly Ser Trp Ala Ala Arg Val Asn Tyr Ser Glu Cys Gln Glu IleLeu 95 100 105 aat gag gag aaa aaa agc aag gtg cac tac cat gtc gca gtcatc atc 447 Asn Glu Glu Lys Lys Ser Lys Val His Tyr His Val Ala Val IleIle 110 115 120 aac tac ctg ggc cac tgt atc tcc ctg gtg gcc ctc ctg gtggcc ttt 495 Asn Tyr Leu Gly His Cys Ile Ser Leu Val Ala Leu Leu Val AlaPhe 125 130 135 gtc ctc ttt ctg cgg ctc agg cca ggc tgc acc cat tgg ggtgac cag 543 Val Leu Phe Leu Arg Leu Arg Pro Gly Cys Thr His Trp Gly AspGln 140 145 150 gca gat gga gcc ctg gag gtg ggg gct cca tgg agt ggt gcccca ttt 591 Ala Asp Gly Ala Leu Glu Val Gly Ala Pro Trp Ser Gly Ala ProPhe 155 160 165 170 cag gtt cga agg agc atc cgg tgc ctg cga aac atc atccac tgg aac 639 Gln Val Arg Arg Ser Ile Arg Cys Leu Arg Asn Ile Ile HisTrp Asn 175 180 185 ctc atc tcc gcc ttc atc ctg cgc aac gcc acc tgg ttcgtg gtc cag 687 Leu Ile Ser Ala Phe Ile Leu Arg Asn Ala Thr Trp Phe ValVal Gln 190 195 200 cta acc atg agc ccc gag gtc cac cag agc aac gtg ggctgg tgc agg 735 Leu Thr Met Ser Pro Glu Val His Gln Ser Asn Val Gly TrpCys Arg 205 210 215 ttg gtg aca gcc gcc tac aac tac ttc cat gtg acc aacttc ttc tgg 783 Leu Val Thr Ala Ala Tyr Asn Tyr Phe His Val Thr Asn PhePhe Trp 220 225 230 atg ttc ggc gag ggc tgc tac ctg cac aca gcc atc gtgctc acc tac 831 Met Phe Gly Glu Gly Cys Tyr Leu His Thr Ala Ile Val LeuThr Tyr 235 240 245 250 tcc act gac cgg ctg cgc aaa tgg atg ttc atc tgcatt ggc tgg ggt 879 Ser Thr Asp Arg Leu Arg Lys Trp Met Phe Ile Cys IleGly Trp Gly 255 260 265 gtg ccc ttc ccc atc att gtg gcc tgg gcc att gggaag ctg tac tac 927 Val Pro Phe Pro Ile Ile Val Ala Trp Ala Ile Gly LysLeu Tyr Tyr 270 275 280 gac aat gag aag tgc tgg ttt ggc aaa agg cct ggggtg tac acc gac 975 Asp Asn Glu Lys Cys Trp Phe Gly Lys Arg Pro Gly ValTyr Thr Asp 285 290 295 tac atc tac cag ggc ccc atg atc ctg gtc ctg ctgatc aat ttc atc 1023 Tyr Ile Tyr Gln Gly Pro Met Ile Leu Val Leu Leu IleAsn Phe Ile 300 305 310 ttc ctt ttc aac atc gtc cgc atc ctc atg acc aagctc cgg gca tcc 1071 Phe Leu Phe Asn Ile Val Arg Ile Leu Met Thr Lys LeuArg Ala Ser 315 320 325 330 acc acg tct gag acc att cag tac agg aag gctgtg aaa gcc act ctg 1119 Thr Thr Ser Glu Thr Ile Gln Tyr Arg Lys Ala ValLys Ala Thr Leu 335 340 345 gtg ctg ctg ccc ctc ctg ggc atc acc tac atgctg ttc ttc gtc aat 1167 Val Leu Leu Pro Leu Leu Gly Ile Thr Tyr Met LeuPhe Phe Val Asn 350 355 360 ccc ggg gag gat gag gtc tcc cgg gtc gtc ttcatc tac ttc aac tcc 1215 Pro Gly Glu Asp Glu Val Ser Arg Val Val Phe IleTyr Phe Asn Ser 365 370 375 ttc ctg gaa tcc ttc cag ggc ttc ttt gtg tctgtg ttc tac tgt ttc 1263 Phe Leu Glu Ser Phe Gln Gly Phe Phe Val Ser ValPhe Tyr Cys Phe 380 385 390 ctc aat agt gag gtc cgt tct gcc atc cgg aagagg tgg cac cgg tgg 1311 Leu Asn Ser Glu Val Arg Ser Ala Ile Arg Lys ArgTrp His Arg Trp 395 400 405 410 cag gac aag cac tcg atc cgt gcc cga gtggcc cgt gcc atg tcc atc 1359 Gln Asp Lys His Ser Ile Arg Ala Arg Val AlaArg Ala Met Ser Ile 415 420 425 ccc acc tcc cca acc cgt gtc agc ttt cacagc atc aag cag tcc aca 1407 Pro Thr Ser Pro Thr Arg Val Ser Phe His SerIle Lys Gln Ser Thr 430 435 440 gca gtc tgagctggca ggtcatggag cagcccccaaagagctgtgg ctggggggat 1463 Ala Val gacggccagg ctccctgacc accctgcctgtggaggtgac ctgttaggtc tcatgcccac 1523 tcccccagga gcagctggca ctgacagcctgggggggccg ctctccccct gcagccgtg 1582 15 444 PRT Homo sapiens 15 Met GlyGly His Pro Gln Leu Arg Leu Val Lys Ala Leu Leu Leu Leu 1 5 10 15 GlyLeu Asn Pro Val Ser Ala Ser Leu Gln Asp Gln His Cys Glu Ser 20 25 30 LeuSer Leu Ala Ser Asn Ile Ser Gly Leu Gln Cys Asn Ala Ser Val 35 40 45 AspLeu Ile Gly Thr Cys Trp Pro Arg Ser Pro Ala Gly Gln Leu Val 50 55 60 ValArg Pro Cys Pro Ala Phe Phe Tyr Gly Val Arg Tyr Asn Thr Thr 65 70 75 80Asn Asn Gly Tyr Arg Glu Cys Leu Ala Asn Gly Ser Trp Ala Ala Arg 85 90 95Val Asn Tyr Ser Glu Cys Gln Glu Ile Leu Asn Glu Glu Lys Lys Ser 100 105110 Lys Val His Tyr His Val Ala Val Ile Ile Asn Tyr Leu Gly His Cys 115120 125 Ile Ser Leu Val Ala Leu Leu Val Ala Phe Val Leu Phe Leu Arg Leu130 135 140 Arg Pro Gly Cys Thr His Trp Gly Asp Gln Ala Asp Gly Ala LeuGlu 145 150 155 160 Val Gly Ala Pro Trp Ser Gly Ala Pro Phe Gln Val ArgArg Ser Ile 165 170 175 Arg Cys Leu Arg Asn Ile Ile His Trp Asn Leu IleSer Ala Phe Ile 180 185 190 Leu Arg Asn Ala Thr Trp Phe Val Val Gln LeuThr Met Ser Pro Glu 195 200 205 Val His Gln Ser Asn Val Gly Trp Cys ArgLeu Val Thr Ala Ala Tyr 210 215 220 Asn Tyr Phe His Val Thr Asn Phe PheTrp Met Phe Gly Glu Gly Cys 225 230 235 240 Tyr Leu His Thr Ala Ile ValLeu Thr Tyr Ser Thr Asp Arg Leu Arg 245 250 255 Lys Trp Met Phe Ile CysIle Gly Trp Gly Val Pro Phe Pro Ile Ile 260 265 270 Val Ala Trp Ala IleGly Lys Leu Tyr Tyr Asp Asn Glu Lys Cys Trp 275 280 285 Phe Gly Lys ArgPro Gly Val Tyr Thr Asp Tyr Ile Tyr Gln Gly Pro 290 295 300 Met Ile LeuVal Leu Leu Ile Asn Phe Ile Phe Leu Phe Asn Ile Val 305 310 315 320 ArgIle Leu Met Thr Lys Leu Arg Ala Ser Thr Thr Ser Glu Thr Ile 325 330 335Gln Tyr Arg Lys Ala Val Lys Ala Thr Leu Val Leu Leu Pro Leu Leu 340 345350 Gly Ile Thr Tyr Met Leu Phe Phe Val Asn Pro Gly Glu Asp Glu Val 355360 365 Ser Arg Val Val Phe Ile Tyr Phe Asn Ser Phe Leu Glu Ser Phe Gln370 375 380 Gly Phe Phe Val Ser Val Phe Tyr Cys Phe Leu Asn Ser Glu ValArg 385 390 395 400 Ser Ala Ile Arg Lys Arg Trp His Arg Trp Gln Asp LysHis Ser Ile 405 410 415 Arg Ala Arg Val Ala Arg Ala Met Ser Ile Pro ThrSer Pro Thr Arg 420 425 430 Val Ser Phe His Ser Ile Lys Gln Ser Thr AlaVal 435 440

That which is claimed is:
 1. An isolated mammalian G protein-coupledcorticotropin-releasing factor (CRF) receptor protein, wherein saidprotein is encoded by DNA that hybridizes under suitable stringency tothe complement of polynucleotide sequences set forth in SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9 or SEQ ID NO: 14, so as to allow identification ofsequences having at least 50% nucleic acid identity with respect to thereference polynucleotide sequences; wherein said receptor protein bindsCRF; and wherein said protein is at least about 70% pure (by weight oftotal proteins).
 2. The isolated protein according to claim 1 havingsufficient binding affinity for CRF such that concentrations of lessthan or equal to 10 nanomolar CRF occupy greater than or equal to 50% ofthe binding sites of said receptor protein.
 3. The isolated proteinaccording to claim 1, wherein said protein is encoded by DNA having atleast 60% nucleic acid identity with respect to the referencepolynucleotide sequences.
 4. The isolated protein according to claim 1,wherein said protein is encoded by DNA having at least 70% nucleic acididentity with respect to the reference polynucleotide sequences.
 5. Theisolated protein according to claim 1, wherein said protein is encodedby DNA having at least 80% nucleic acid identity with respect to thereference polynucleotide sequences.
 6. The isolated protein according toclaim 1, wherein said protein is encoded by DNA having at least 90%nucleic acid identity with respect to the reference polynucleotidesequences.
 7. The isolated protein according to claim 1 having the aminoacid sequence set forth in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10 orSEQ ID NO:
 15. 8. The isolated protein according to claim 1 having aradioactive labelling element attached thereto.
 9. The isolated proteinaccording to claim 1, wherein said isolated protein is a recombinantprotein.
 10. A composition comprising an isolated protein according toclaim
 1. 11. An immunogenic fragment of an isolated mammalian Gprotein-coupled corticotropin-releasing factor (CRF) receptor protein;wherein said protein is encoded by DNA that hybridizes under suitablestringency to the complement of polynucleotide sequences set forth inSEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO: 14, so as to allowidentification of sequences having at least 50% nucleic acid identitywith respect to the reference polynucleotide sequences; wherein saidreceptor protein binds CRF; and wherein said protein is at least about70% pure (by weight of total proteins).
 12. An antibody generatedagainst a polypeptide according to claim
 11. 13. A substantially purepolypeptide comprising at least 15 contiguous amino acids of the aminoacid sequence set forth in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10 orSEQ ID NO: 15; wherein said polypeptide is at least about 70% pure (byweight of total proteins).
 14. The polypeptide according to claim 13,wherein a residue selected from the group consisting of tyrosine,cysteine, lysine, glutamic acid and aspartic acid has been attached by apeptide bond to the carboxyl terminus of said polypeptide.
 15. Anisolated mammalian G protein-coupled corticotropin-releasing factor(CRF) receptor protein, wherein said protein is encoded by DNA thathybridizes to the complement of polynucleotide sequences set forth inSEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO: 14, underhybridization conditions comprising a temperature of about 42° C., aformamide concentration of about 20% and a salt concentration of about0.6 M NaCl, followed by wash conditions comprising a temperature ofabout 42-50° C. and a salt concentration of about 0.3 M NaCl; whereinsaid receptor protein binds CRF; and wherein said protein is at leastabout 70% pure (by weight of total proteins).
 16. The isolated proteinaccording to claim 15, wherein said isolated protein is a recombinantprotein.
 17. An isolated mammalian G protein-coupledcorticotropin-releasing factor (CRF) receptor protein, wherein saidprotein is encoded by DNA that hybridizes to the complement ofpolynucleotide sequences set forth in SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9 or SEQ ID NO: 14, under hybridization conditions comprising atemperature of about 42° C., a formamide concentration of about 50%, anda salt concentration of about 5×SSPE, followed by wash conditionscomprising a temperature of about 65° C. and a salt concentration ofabout 0.2×SSPE; wherein said receptor protein binds CRF; and whereinsaid protein is at least about 70% pure (by weight of total proteins).18. The isolated protein according to claim 17, wherein said isolatedprotein is a recombinant protein.
 19. A diagnostic kit for assaying forthe presence in biological fluids of CRF-R protein, CRF-R proteinanalogs, and/or CRF-R fragments, said kit comprising: (a) an isolatedmammalian G protein-coupled corticotropin-releasing factor (CRF)receptor protein according to claim 1, and/or (b) one or more antibodiesgenerated against said protein or immunologic fragment thereof.